Multiplex q-pcr arrays

ABSTRACT

This invention provides methods and systems for measuring the concentration of multiple nucleic acid sequences in a sample. The nucleic acid sequences in the sample are simultaneously amplified, for example, using polymerase chain reaction (PCR) in the presence of an array of nucleic acid probes. The amount of amplicon corresponding to the multiple nucleic acid sequences can be measured in real-time during or after each cycle using a real-time microarray. The measured amount of amplicon produced can be used to determine the original amount of the nucleic acid sequences in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of, and claims priority toand the benefit of, co-pending U.S. patent application Ser. No.11/829,861, filed Jul. 27, 2007, now U.S. Pat. No. 8,______, whichapplication claimed the priority and the benefit of then co-pending U.S.Provisional Application No. 60/834,051, filed Jul. 28, 2006, each ofwhich applications is incorporated herein by reference in its entirety.This application is also related to U.S. patent application Ser. No.11/758,621 filed Jun. 5, 2007 and U.S. patent application Ser. No.11/844,996 filed Aug. 24, 2007.

BACKGROUND OF THE INVENTION

Nucleic acid target amplification assays such as polymerase chainreaction process (PCR), in principle, amplify and replicate specificsequences of nucleic acids of a DNA template in vitro. These assays havebecome a powerful tool in molecular biology and genomics, since they canincrease the number of copies of target molecules with greatspecificity. It is of great interest to efficiently multiplex theamplification process and thus allow for multiple target amplificationand quantification.

Currently, various homogeneous (closed-tube) assays are available forPCR. These assays detect the target amplicons (i.e., Quantitative PCR orQPCR). Nevertheless, the number of different nucleic acid sequences thatcan be simultaneously amplified and detected is very limited. Typically,an individual reaction chamber (or well) where the target is amplifiedand detected contains only a single amplicon. Multiplexed QPCR, definedas the process of amplifying and detecting a plurality of nucleic acidsequences simultaneously in a single reaction chamber, is generallypractical only for a small number of amplicons.

PCR relies on an enzymatic replication process in each of itstemperature-regulated cycles (typically, 30-40). A PCR cycle typicallyconsists of three distinct phases: denaturing, annealing, and extension.Ideally, at the end of the extension phase, there are twice as manydouble-stranded target DNA fragments as there were at the beginning ofthe cycle. This implies an exponential growth of the amount of thetarget DNA as one proceeds through the cycles. However, practical issuesaffect the replication process adversely and the efficiency of PCR,defined as the probability of generating a replica of each templatemolecule, is usually smaller than the desired factor of two.

Quantification of the amplified targets in PCR is typically based onmeasuring the light intensity emanating from fluorescent reportermolecules incorporated into the double-stranded DNA copies of thetarget. The measured light intensity is an indication of the actualnumber of the amplified targets. Some of the commonly used probes areSYBR Green, hybridization, and TaqMan probes. SYBR Green I is a dyewhose fluorescence increases significantly upon binding to(intercalating) double-stranded DNA. SYBR Green is non-discriminatoryand will bind to non-specific byproducts of PCR (such as theprimer-dimers). For this reason, special attention needs to be paid tooptimizing the conditions of PCR with SYBR Green reporters.Hybridization probes are specific to the target DNA sequence. Ahybridization probe typically consists of two short probe sequences, onelabeled with a fluorescence resonance energy transfer donor and theother with an acceptor dye. The two probe sequences can be designed suchthat they hybridize next to each other on the target sequence; theco-location of the donor and the acceptor initiates energy transfer and,therefore, the change in their respective light intensities indicatessuccessful replication of the target. The TaqMan probes are alsospecific to the target sequence, and are designed so that they contain afluorescent dye in the vicinity of a quenching dye. Where the TaqManprobe hybridizes to the template, and the template is replicated, theTaqMan probe is degraded by the exo activity of the polymerase,separating the fluorescent dye from the quenching dye, resulting in anincreased fluorescent signal.

In Q-PCR, fluorescent signal is measured at the end of each temperaturecycle. The measured light intensities create a reaction profile, whichcan be plotted against the number of cycles and used to determine theconcentration of the nucleic acid sequence that was amplified.

Attempts at employing QPCR for the simultaneous amplification anddetection of many target amplicons in a single well are plagued withpractical issues that present obstacles to achieving a truly multiplexedQ-PCR. A common approach used is to divide the biological sample ofinterest into equal-sized quantities which are then mechanicallydelivered into separate wells (typically, 96, 192, or 384). This type ofsample splitting reduces the amount of material in each individualamplification well, creating issues of sample size, and necessitatingprecise sample distribution across the wells.

On the other hand, high-throughput screenings of multiple targetanalytes in biological samples is typically obtained by exploiting theselective binding and interaction of recognition probes inmassively-parallel affinity-based biosensors, such as microarrays. Geneexpression microarrays, for example are widely used microarrayplatforms. These systems measure the expression level of thousands ofgenes simultaneously.

To increase the quality of microarray data, real-time microarray(RT-μArray) systems have been developed. (see U.S. patent applicationSer. No. 11/758,621). These systems can evaluate the abundance of aplurality of target analytes in the sample by real-time detection oftarget-probe binding events. To achieve this, RT-μArrays employ adetection scheme that is a major departure from the techniques typicallyused in conventional fluorescent-based microarrays and other extrinsicreporter-based biosensors assays. In the latter, the detection ofcaptured analytes is carried out after the hybridization(incubation)-step. This is due to the characteristics of the assays usedtherein, which require removing the solution during the fluorescent andreporter intensity measurements that are done either by scanning and/orvarious other imaging techniques. This limitation is due in part to thehigh concentration in solution of unbound labeled species which canoverwhelm the target-specific signal from the captured targets.Furthermore, when the hybridization is ceased and the solution is takenaway from array surface, washing artifacts typically occur which canmake the analysis of the data challenging.

Thus, while Q-PCR provides a convenient and accurate method formeasuring the amount of nucleic acid sequences in a sample, it islimited to a single sequence or a small number of sequences in a singlefluid volume. And while microarray technology provides for measuringover hundreds of thousands of sequences simultaneously, conventionalarrays are not amenable to the type of detection required for practicalmultiplexed Q-PCR. Thus there exists a strong need for methods andsystems for performing multiplex Q-PCR to accurately measure thepresence and/or amount of multiple nucleic acid sequences in a singlefluid volume in a single amplification reaction.

SUMMARY OF THE INVENTION

One aspect of the invention is a method comprising; performing a nucleicacid amplification on two or more nucleotide sequences to produce two ormore amplicons in a fluid wherein the array comprises a solid surfacewith a plurality of nucleic acid probes at independently addressablelocations; and measuring the hybridization of the amplicons to the twoor more nucleic acid probes while the fluid is in contact with the arrayto obtain an amplicon hybridization measurement. In some embodiments theinvention further comprises using the amplicon hybridization measurementto determine the concentration of the amplicons in the fluid. In someembodiments the invention further comprises using the ampliconhybridization measurement to determine the original amount of nucleotidesequences.

In some embodiments the concentration of amplicon is measured during orafter some, but not all of the amplification cycles. In some embodimentsthe concentration of amplicon is measured during or after each of theamplification cycles. In some embodiments the concentration of ampliconis measured during on average every 2, 3, 4, 5, 6, 7, 8, 9 or 10amplification cycles. In some embodiments the nucleic acid amplificationis polymerase chain reaction (PCR), and an amplification cyclecorresponds with a temperature cycle. In some embodiments at least sometemperature cycles comprise (a) a probe hybridization phase at onetemperature, and (b) primer annealing phase at a higher temperature thanthe probe hybridization temperature. In some embodiments at least sometemperature cycles comprise 4 or more temperature phases, wherein one ormore of the phases is a probe hybridization phase wherein thehybridization of amplicons to the nucleic acid probes is measured. Insome embodiments the temperature is changed during probe hybridizationphase. In some embodiments the method comprises two or morehybridization phases, carried out at different temperatures. In someembodiments at least some temperature cycles comprise a first denaturingphase, a probe hybridization phase, a second denaturing phase, a primerannealing phase, and an extension phase.

In some embodiments the array is in contact with the fluid during theamplification.

In some embodiments the two or more nucleotide sequences are not onlytwo complementary nucleotide sequences.

In some embodiments the amplification is an isothermal amplification. Insome embodiments the amplification is a linear amplification.

In some embodiments the measuring of hybridization comprises measuringthe kinetics of hybridization of the amplicons to the nucleic acidprobes. In some embodiments the measuring of the kinetics ofhybridization comprises measuring a light signal at multiple timepoints.

In some embodiments the amplicons comprise a quencher. In someembodiments primers are used to create the amplicons and the primerscomprise a quencher. In some embodiments one of the primers in a primerpair comprises a quencher. In some embodiments both of the primers in aprimer pair comprise a quencher. In some embodiments quenchers areincorporated into the amplicons as they are formed. In some embodimentsd-NTP's are used to make the amplicons, and one or more of the d-NTP'sused to make the amplicon comprises a quencher.

In some embodiments the amplicon hybridization measurement is performedby measuring fluorescence from fluorescent moieties attached to thesolid surface. In some embodiments the fluorescent moieties arecovalently attached to the nucleic acid probes. In some embodiments thefluorescent moieties are attached to the substrate and are notcovalently attached to the nucleic acid probes. In some embodiments theamplicons comprise quenchers, and the measuring of hybridization isperformed by measuring a decrease in fluorescence due to hybridizationof amplicons to the nucleic acid probes. In some embodiments the arraycomprises at least about 3, 4, 5 10, 50, 100, 100, 1000, 10,000,100,000, or 1,000,000 nucleic acid probes. In some embodiments thenucleic acid probe comprises a PNA or LNA probe.

One aspect of the invention is a method comprising: (a) providing anarray comprising a solid support having a surface and a plurality ofdifferent probes, the different probes immobilized to the surface atdifferent addressable locations, each addressable location comprising afluorescent moiety; (b) performing PCR amplification on a samplecomprising a plurality of nucleotide sequences; the PCR amplificationcarried out in a fluid, wherein: (i) a PCR primer for each nucleic acidsequence comprises a quencher; and (ii) the fluid is in contact with theprobes, whereby amplified molecules can hybridize with probes, therebyquenching signal from the fluorescent moiety; (c) detecting the signalsfrom the fluorescent moieties at the addressable locations over time;(d) using the signals detected over time to determine the amount ofamplified molecules in the fluid; and (e) using the amount of amplifiedmolecules in the fluid to determine the amount of the nucleotidesequences in the sample.

In some embodiments the determining of the amount of amplified moleculesis performed during or after multiple temperature cycles of the PCRamplification. In some embodiments more than one PCR primer for eachnucleic acid sequence comprises a quencher. In some embodimentsdetecting of the signals from the fluorescent moieties at theaddressable locations over time comprises measuring the rate ofhybridization of the amplified molecules with the probes.

In some embodiments the sample comprises messenger RNA or nucleotidesequences derived from messenger RNA, and the determination of theamount of nucleic acid sequence in the sample is used to determine thelevel of gene expression in a cell or group of cells from which thesample was derived. In some embodiments the sample comprises genomic DNAor nucleotide sequences derived from genomic DNA, and the determinationof the amount of nucleic acid sequence in the sample is used todetermine the genetic makeup of a cell or group of cells from which thesample was derived.

In some embodiments two or more PCR primers corresponding to two or moredifferent nucleotide sequences have different quenchers. In someembodiments two or more different addressable locations comprisedifferent fluorescent moieties. In some embodiments the differentquenchers and/or different fluorescent moieties are used to determinecross-hybridization.

One aspect of the invention is a diagnostic test for determining thestate of health of an individual comprising performing a methoddescribed herein on a sample from such individual.

One aspect of the invention is a method comprising measuring the amountof 10 or more amplicons corresponding to 10 or more different nucleotidesequences in a single fluid volume during or after multipleamplification cycles to determine amplicon amount-amplification cyclevalues, and using the amplicon amount-amplification cycle values todetermine the presence or amount of the 10 or more nucleotide sequencesin a sample. In some embodiments 20 or more amplicons corresponding to20 or more different nucleotide sequences are used to determine theamount of 20 or more nucleotide sequences. In some embodiments 50 ormore amplicons corresponding to 50 or more different nucleotidesequences are used to determine the amount of 50 or more nucleotidesequences.

In some embodiments the multiple amplification cycles comprise about10-40 amplification cycles. In some embodiments the amount of the 10 ormore amplicons is measured in real-time. In some embodiments amount ofthe 10 or more amplicons is measured by measuring the kinetics ofbinding of the amplicons to nucleic acid probes. In some embodiments theamount of the 10 or more amplicons is measured by measuring thequenching of fluorescence.

One aspect of the invention is system comprising: a PCR amplificationreaction chamber capable of receiving: a substrate comprising a surfacewith an array of nucleic acid probes at independently addressablelocations, and a fluid to be held contact with the substrate, the fluidcomprising a nucleic acid sample comprising multiple nucleotidesequences, primers, and enzymes; a temperature controller capable ofcarrying out multiple PCR temperature phases and temperature cyclescomprising: a heating and cooling module for raising and lowering thetemperature of the fluid and/or the substrate; and a temperature sensor;and a detector capable of detecting light signals as a function of timefrom the independently addressable locations on, the substrate withinthe chamber at a specific phase or phases during or after a plurality oftemperature cycles while the fluid is in contact with the substrate.

In some embodiments the invention further comprises: (d) an analysisblock comprising a computer and software capable of determining theamounts of amplified products hybridized to the array of probes usingthe detected light as a function of time, and of determining the amountsof multiple nucleotide sequences in a sample using the amounts ofamplified products determined during or after a plurality of temperaturecycles.

In some embodiments the detector comprises a photodiode, a CCD array, ora CMOS array.

In some embodiments the detector is in contact with the substrate, anddifferent areas of the detector correspond to different detectablelocations. In some embodiments the detector is optically coupled to thesubstrate with lenses and/or waveguides.

In some embodiments the heating and cooling module is capable of raisingor lowering the temperature at a rate of greater than 5° C. per sec. Insome embodiments the heating and cooling module is capable of raising orlowering the temperature at a rate of greater than 10° C. per sec. Insome embodiments the heating and cooling module is capable of raising orlowering the temperature at a rate of greater than 20° C. per sec. Insome embodiments the temperature sensor is capable of measuringtemperature at an accuracy of 0.1° C.

In some embodiments the system comprises a microfluidic device havingmultiple arrays, each of the multiple arrays is in a chamber which canhold the fluid to be held in contact with the substrate. In someembodiments at least some of the multiple arrays are addressed withdifferent temperature profiles. In some embodiments the microfluidicdevice has about 4, 5, 6, 7, 8, 9, or 10 arrays.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an embodiment of the invention wherein primers (P1,P2) with quenchers (Q1, Q2) are incorporated into amplicons in anamplification reaction, allowing the amplicons to be detected inreal-time by hybridization with probes on an array resulting inquenching of the fluorescence of fluorescent moieties (F1, F2) attachedto the probes. The measurement of amplicons after multiple amplificationcycles can be used to determine the concentration of the starting targetnucleotide sequence (A and/or B).

FIG. 2 shows how, for the embodiment shown in FIG. 1, primers (P1, P2)with quenchers (Q1, Q2) can, in some instances, hybridize to the 3′ endof amplicons that are hybridized to probes (C′, D′) on the array.

FIG. 3 illustrates an embodiment of the invention in which the probes(P1, P2) and amplicons are designed such that a primer with a quencher(Q1, Q2) hybridizes to the 3′ end of an amplicon which is hybridized toa probe on an array, resulting in quenching of the fluorescence of afluorescent moiety (F1, F2) attached to the probe.

FIG. 4 illustrates an embodiment of the invention in which quenchers (Q)are incorporated into amplicons during the amplification reaction byusing a d-NTP containing a quencher in the amplification. Thequencher-labeled amplicons are detected in real-time by hybridization toprobes on an array resulting in quenching of fluorescent moieties (F)attached to the array. The measurement of amplicons after multipleamplification cycles can be used to determine the concentration of thestarting target nucleotide sequence (A and/or B).

FIG. 5 shows how, in some embodiments of the invention, the fluorescentmoieties (F) are bound to the surface of the array, but are notcovalently bound to the probes. The fluorescent moieties can be bounddirectly or attached to another molecule bound to the surface. Thesurface bound fluorescent moieties can be quenched upon hybridization ofamplicons containing quenchers (Q) to probes on the array, allowing themeasurement of amplicon hybridization in real time.

FIG. 6 shows conventional detection in the dry state after completingincubation at time t₁ and the uncertainty associated with it.

FIG. 7 shows that in real-time microarray systems of the presentinvention, multiple measurement of the number of captured amplicons canbe carried out, providing more accurate information about binding.

FIG. 8 shows a block diagram of the errors associated with conventionalDNA microarrays that require washing and drying before detection.

FIG. 9 shows a real-time array of the present invention where the probesare labeled with fluorescent moieties.

FIG. 10 shows a real-time array of the present invention where theprobes are labeled with fluorescent moieties, the amplicons are labeledwith quenchers, and the fluorescent intensity on various spots can beused to measure the amount of amplicon specifically bound to probe.

FIG. 11 shows an embodiment of a temperature cycle of the presentinvention where the temperature cycle has 5 phases.

FIG. 12 shows a real-time microarray system where the detection systemcomprises a sensor array in intimate proximity of the capturing spots.

FIG. 13 shows a block diagram of a real-time microarray system of thepresent invention.

FIG. 14 shows an example of a real-time array system where binding ofBHQ2 quencher-labeled cDNA molecules are detected using a fluorescentlaser-scanning microscope.

FIG. 15 shows an example of a layout of a 6×6 DNA array.

FIG. 16 shows a few samples of real-time measurements in a microarrayexperiment where control target analytes are added to the systemdemonstrating the measurement of hybridization in real time.

FIGS. 17-20 each show real-time data for 4 different spots with similaroligonucleotide capturing probes. A target DNA analyte is introduced inthe system at time zero and quenching (reduction of signal) occurs uponhybridization.

FIG. 21 shows the signals measured during two real-time experimentswherein a target analyte (target 2) is applied to the microarray, at 2ng and at 0.2 ng.

FIG. 22 shows the signal measured in a real-time array, and the fit ofthe data to an algorithm for determining amount of analyte present inthe fluid, where 80 ng of the target is applied to the array in 50 μl.

FIG. 23 shows signal measured in a real-time array, and the fit of thedata to an algorithm for determining amount of analyte present in thefluid, where 16 ng of the target is applied to the array in 50 μl.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The methods, devices, and systems disclosed herein relate to thedetection and measurement of multiple nucleotide sequences in a sampleby performing real-time measurements of amplicon binding events tonucleic acid probes bound to microarrays. The methods and systemsdescribed herein utilize real-time microarray (RT-μArray) systems.Real-time microarray systems are described in the U.S. patentapplication Ser. No. 11/758,621 which is incorporated herein byreference.

One aspect of the invention involves performing a PCR amplification in asolution in contact with a probe array, where the solution containsnucleotide sequences of interest. The probes are bound to the array atindependently addressable locations and have fluorescent moieties. Theprobes are designed to specifically hybridize with amplicons generatedby the PCR, and the amplicons are designed such that hybridization ofthe amplicon to the probe results in quenching of the fluorescentsignals from the probes. During each cycle of PCR, hybridization betweenamplicon and probe is detected, and the denaturing step of PCR releasesthe amplicons for continued amplification. Preferably, the hybridizationis measured in real-time during hybridization of amplicons to probes,producing kinetic measurements that can be used to determine the amountof amplicon in solution. Performing the measurement over many cyclesallows the measurement of the build-up of amplicon as a function ofamplification cycle, which can be extrapolated back in order todetermine the concentration of the nucleotide sequence in the sample.The determination of the amount of nucleotide sequence by ampliconbuild-up is analogous to a CT measurement using Q-PCR, but the presentinvention, being in an array format, allows for a multiplex assay.

In one aspect of the invention, rather than having the fluorescentmoieties that are quenched by the amplicons attached to the probe, thefluorescent moieties are attached to the surface within theindependently addressable location of the probe. While the fluorescentmoiety is not covalently bound to the probe, we have found that thefluorescence can still be quenched upon hybridization. This aspectallows for the measurement of amplicon-probe binding without the needfor synthesizing fluorescently labeled probes.

The real-time measurement of the kinetics of multiple binding eventsallows for an accurate and sensitive determination of bindingcharacteristics or of analyte concentrations for multiple speciessimultaneously. Real-time measurements also allow for the rapiddetermination of the amount of analyte present in a fluid, because themeasurement can be made without waiting for saturation of binding. Oneaspect of present invention is the evaluation of the abundance ofnucleotide sequences in a sample by the real-time detection ofamplicon-probe binding events of amplicons derived from the nucleotidesequences. In certain embodiments, RT-μArray detection systems measurethe concentration of the amplicons by analyzing the binding rates and/orthe equilibrium concentration of the captured amplicons in a pluralityof spots.

In some embodiments, the amplification method that is used isquantitative-PCR or Q-PCR. In Q-PCR, a PCR reaction is carried out toamplify a nucleotide sequence. The amount of amplicon produced ismeasured at the end of each amplification cycle. After a number ofamplification cycles, the amount of nucleic acid in the original samplecan be determined by analyzing the build-up of amplicon over the numberof amplification cycles. Q-PCR allows for the determination of thepresence or the amount very small amounts of sample, in some cases, downto the detection of a single molecule. While Q-PCR is a valuabletechnique for measuring nucleic acid sequences, the ability to measuremultiple sequences at a time in a single fluid with Q-PCT reaction islimited. The present invention utilizes an array of multiple nucleicacids, thus allowing for the measurement of the presence or amount ofmultiple nucleic acid sequences in the same sample during the same Q-PCRamplification reaction. In some embodiments, the present inventionallows for the determination of more than about 3, 5, 10, 100, 1000,10,000, 100,000, 1,000,000 or more nucleic acid sequences in the samesample during the same amplification reaction.

The term “quantitative-PCR” or “Q-PCR” as used herein to refer to theprocess that is also referred to outside of this application as“real-time PCR”. The term “Q-PCR” as used herein encompasses both thequalitative and quantitative determination of nucleic acid sequences.Q-PCR involves the measurement of the amount of amplicon as a functionof amplification cycle, and using this information in order to determinethe amount of the nucleic acid sequence corresponding to the ampliconthat was present in the original sample. The Q-PCR process can bedescribed in the following manner. A PCR reaction is carried out with apair of primers designed to amplify a given nucleic acid sequence in asample. The appropriate enzymes and dNTP's are added to the reaction,and the reaction is subjected to a number of amplification cycles. Theamount of amplicon generated from each cycle is detected, but in theearly cycles, the amount of amplicon can be below the detectionthreshold. The amplification can be seen as occurring in two phases, anexponential phase, followed by a non-exponential plateau phase. Duringthe exponential phase, the amount of PCR product approximately doublesin each cycle. As the reaction proceeds, however, reaction componentsare consumed, and ultimately one or more of the components becomeslimiting. At this point, the reaction slows and enters the plateau phase(generally between cycles 28-40). Initially, the amount of ampliconremains at or below background levels, and increases are not detectable,even though amplicon product accumulates exponentially. Eventually,enough amplified product accumulates to yield a detectable signal. Thecycle number at which this occurs is called the threshold cycle, or CT.Since the CT value is measured in the exponential phase when reagentsare not limited, Q-PCR can be used to reliably and accurately calculatethe initial amount of template present in the reaction. The CT of areaction is determined mainly by the amount of nucleic acid sequencecorresponding to amplicon present at the start of the amplificationreaction. If a large amount of template is present at the start of thereaction, relatively few amplification cycles will be required toaccumulate enough product to give a fluorescent signal above background.Thus, the reaction will have a low, or early, CT. In contrast, if asmall amount of template is present at the start of the reaction, moreamplification cycles will be required for the fluorescent signal to riseabove background. Thus, the reaction will have a high, or late, CT. Thepresent invention allows for the measurement of the accumulation ofmultiple amplicons in a single fluid in a single amplification reaction,and thus the determination of the amount of multiple nucleic acidsequences in the same sample with the methodology of Q-PCR describedabove.

In some embodiments the Q-PCR can incorporate a blocker. For example, asequence that is complementary to the nucleotide sequence beingamplified can act as a blocker by blocking the synthesis of theamplicon. Blockers that are sensitive to small changes in nucleotidesequence can be used, for example, for single nucleotide polymorphism(SNP) determination. In some embodiments, the blocker is made from anon-natural nucleic acid with different, for example, stronger,hybridization characteristics. In some embodiments the blocker is madefrom locked nucleic acid (LNA) or protein nucleic acid (PNA).

In the current invention, the amplification is carried out in a fluidthat is in contact with an array comprising multiple nucleic acidsprobes, each nucleic acid probe attached to an independently addressablelocation. An independently addressable location is a region of the arraythat can be addressed by a detector to obtain information about ampliconbinding to that specific region. The multiple amplicons that aregenerated can each bind specifically to one or more nucleic acid probesbound to the array, and the rate of binding of the amplicons to thenucleic acids is measured in real-time to determine the amount of theamplicon in the fluid. As with Q-PCR, the sample can be subjected tomultiple amplification cycles, and the amount of amplicon during orafter the cycles can be measured. At the end of multiple amplificationcycles, the build-up of amplicon as a function of amplification cyclecan be determined, and can be used to determine the presence or amountof the nucleic acid sequences present in the original sample. Thismethod allows the measurement of the presence or amount of tens tohundreds to millions of different nucleotide sequences in the samesample during a single amplification. This type of analysis is notpractical on conventional microarrays that require hybridizations toreach saturation, require washing and drying of the array beforedetection, and often require a separate labeling step. The real-timearrays of the present invention allow for this multiplex Q-PCR becausethe fluid need not be removed from the array to detect the amplicons,and because the measurement of the amount of amplicon can be carried outrapidly without waiting for saturation of binding.

In some embodiments of the present invention, fluorescent resonanceenergy transfer (FRET) and/or quenching of fluorescence is used in orderto determine the amount of amplicon present in the solution. Forexample, primers in a PCR amplification reaction are labeled with aquencher, and the nucleic acid probes on the array are labeled with afluorescent moiety that is quenched upon binding of the amplicon to thenucleic acid probe. As the PCR amplification proceeds, the quenchers areincorporated into the amplicons formed in the amplification. During orafter the temperature cycles of the PCR reaction, the decrease in thefluorescent signal with time from the probes is measured in real-time todetermine the rate of binding of the amplicon to the probe which is usedto determine the amount of amplicon in solution. The values obtained forthe amount of amplicon as a function of amplification cycle can then beused to calculate the presence or amount of the nucleotide sequencescorresponding to the amplicons in the original sample. FIGS. 1-5,described in more detail below, show embodiments of the invention thatutilize FRET and quenching. The use of quenchers rather thanfluorescently labeled amplicons in solution has the advantage that itminimizes the fluorescent background of the solution, thus increasingthe quality of the measurement of the fluorescence at the surface.Because the different nucleic acid probes on the surface are atdifferently addressable locations on the surface, the binding ofmultiple amplicons to multiple probes can be determined in the sameamplification reaction. The invention thus provides a multiplex Q-PCRsystem that can determine the presence or concentration of about 2, 3,4, 5, 6, 8, 10, 20, 30, 50; 100, 200, 500, 1,000, 10,000, 100,000,1,000,000 or more nucleotide sequences in the same fluid in sameamplification reaction.

While a conventional multiplex Q-PCR allows the measurement of a smallnumber of nucleic acid sequences in the same fluid by using dyes ofdifferent wavelengths, it has disadvantages, and is limited to a smallnumber of amplicons. The current method carrying out multiple Q-PCRreactions on a sample, especially for larger numbers of ampliconsgreater than 5 or 8 is to split the sample containing the nucleotidesequence into multiple samples, and perform Q-PCR on all of the samplesin parallel. This method has the disadvantages of uncertainties due tosample splitting, and that a larger sample may be needed in order tohave enough material in each of the split samples. Thus, the presentinvention provides a unique multiplex Q-PCR system allowing for thedetermination of the concentration or presence of about 10 or more,about 20 or more, or about 50 or more nucleotide sequences in the samefluid in the same amplification reaction.

FIG. 1 illustrates an embodiment of the invention in which the primershave attached quenchers such that the quenchers become incorporated intothe amplicons. In FIG. 1, the sample comprises a double stranded DNA,having complementary strands A and B. The sample is amplified using twoprimers of a primer pair (P1 and P2) each primer comprising a quencher(Q1 and Q2). In some embodiments, Q1 and Q2 will be the same quencher.In some embodiments, Q1 and Q2 will be different quenchers. In someembodiments, only one member of the primer pair will have a quencher.The primers are chosen so as to amplify the nucleotide region defined bythe nucleotide region to which each of the primers are complementary oneach of the two strands. The nucleotide sequences of both the A and theB strand are amplified. The double stranded DNA and the primers are thensubjected to multiple temperature cycles in the presence of polymeraseand dNTP's to generate amplified product, or amplicon. Here, either thegenerated double stranded DNA or each of the strands of the generatedDNA can be referred to as amplicons. The primers comprising thequenchers become incorporated into the amplicons, resulting in ampliconswhich contain quenchers. Since the primers are incorporated at the 5′end of the strand that they become incorporated into, the quencher willtend to be at the 5′ end of the amplicon that incorporates the primer.The quencher can be at any location along the primer. The quencher canbe at the 3′ end of the primer, at the 5′ end of the primer, or it canbe attached to nucleotides in between the ends of the primer.

During the amplification, the temperature phases are controlled to allowthe measurement of binding of the generated amplicons to the probes onthe array which is in contact with the fluid containing the amplicons.The amplicons can hybridize to the probes on the array as shown inFIG. 1. FIG. 1 shows, for example probe C′ attached to the surface of anarray. A portion of the sequence of probe C′ is complementary to aportion of the sequence on amplicon C. The length of overlap illustratedin the figures is not necessarily indicative of the region of overlapbetween the probe and the amplicon, which can be, for example, fromseveral bases to thousands of bases long.

Typically, as illustrated in the figures, the region of the ampliconthat corresponds to the primer (and the primers themselves) will not becomplementary to the probe. This design can be useful in preventingprimer from binding the probe, which could result in unwanted quenching,and could result in unwanted nucleic acid strands by priming synthesison the probes.

In this embodiment, probe C′ has a fluorescent moiety attached to it.The fluorescent moiety can be attached at any place along the length ofthe probe. The hybridization of the probe C′ to the amplicon C bringsthe quencher Q2 and the fluorescent moiety, F1 into proximity which canresult in quenching of fluorescence from F1. The quencher Q2 and thefluorescent moiety F1, and their attachment to the amplicon and probecan be modified using methods known in the art in order to increase theinteraction between F1 and Q2 and to increase the effectiveness ofquenching.

The decrease in the fluorescence from F1 thus provides a measure of theamount of binding of amplicon C to probe C′, and can thus be used tomeasure the rate of hybridization of the amplicon C to the probe C′. Themeasured rate of hybridization can be used to determine the amount ofamplicon C in solution. The measurement of the amount of amplicon can bedone during or after some or all of the temperature cycles of theamplification, and the values measured for amount of amplicon as afunction of temperature cycle can be used to determine the originalamount of nucleotide sequence from strand A and or strand B.

In some embodiments, amplicon D, which is complementary to amplicon Ccan concurrently be measured using probe D′, attached to anotherindependently addressable region of the array. By measuring theconcentration of both C and D, complementary amplicon sequences, thereliability of the measurement of amplicon amount, and thus thereliability of the amount of the nucleotide sequences from A and/or Bcan be increased.

FIG. 2 illustrates that in some embodiments, when amplicon C bind toprobe C′ near its 5′ ends, the complementary primer P1 with quencher Q1can hybridize to the 3′ end of the amplicon C. In some embodiments thisbinding of primer can be minimized or prevented by controlling thedesign of the primer, amplicon, and probe or by controlling thestringency conditions such as the temperature. For example, thehybridization temperature can be chosen to allow amplicon to probebinding, but not to allow primer binding. In some embodiments, thecomponents can be selected such that the hybridization of Q1 to C doesnot affect the quenching of the fluorescence at the surface of array. Inother embodiments, the hybridization of Q1 to C can act to furtherquench the fluorescence at the surface of the array. FIG. 2 shows thatprimer P2 with quencher Q2 can analogously hybridize to amplicon D,which is hybridized to probe D′.

FIG. 3 shows an embodiment where the Probe C″ is designed to becomplementary to a nucleotide sequence on amplicon C near the 3′ regionrather than near the 5′ region as in FIGS. 1 and 2. In this embodiment,primer, amplicon, probe and conditions are chosen such that primer P1with quencher Q1 is hybridized to the 3′ end of C. The quencher Q1 onprimer P1 is brought into proximity of the fluorescent moiety F1 onprobe C″, resulting in quenching of fluorescence of F1, and providing ameasure of the concentration of the amplicon C. Here, quencher Q2 whichis near the 5′ end of C does not participate in quenching. In someembodiments both the quenching from both Q1 and Q2 can be used. FIG. 3also shows that the same quenching strategy can be used for thecomplementary amplicon D.

FIG. 4 shows an alternative method of incorporating quenchers into theamplicons. Here, one or more of the dNTP's that will become incorporatedinto the amplicons has a quencher Q attached. The quenchers thus becomeincorporated into the formed amplicons. When the amplicons containingthe quenchers Q bind to the surface, the probes and amplicons aredesigned such that the quenchers interact with and quench thefluorescence of the fluorescent moiety F on the surface bound probe. Theprobe can be complementary to either amplicon C or amplicon D, or thearray could incorporate probes to both amplicons C and D. In someembodiments all of the dNTP's of a single type will carry a quencher. Inother cases, only a fraction of the dNTP's of a single type will carry aquencher, resulting in the statistical incorporation of quenchers intothe amplicons.

FIG. 5 illustrates how, in some embodiments, the fluorescent moiety onthe array is not covalently bound directly to the probes, but is bounddirectly to the surface of the array, or is bound through anotherspecies on the surface of the array. In FIG. 5, amplicons with quenchersQ hybridize to the probes on the array, and the quencher Q is broughtinto proximity of the fluorescent moiety on the surface, resulting inquenching that can be used as a measure of the amount of ampliconshybridized to the probes. This aspect of the invention can be useful inthat having the fluorescent moiety attached directly to the surface cansimplify manufacturing, which can improve costs, and improve thereliability of the system. For example, in some embodiments, one mightbe required to synthesize fluorescently labeled probe for each spotwhich can be costly, but by attaching the fluorescent group directly tothe surface, no fluorescently labeled probes need be synthesized.

While the embodiments in FIGS. 1-5 describe quenchers and fluorescentmoieties, it would be understood by persons of skill in the art that inthe above embodiments, any members of a FRET pair could be incorporatedinto the primers, probes, and/or amplicons in the same manner. The useof quenchers on the species in solution has the advantage that thesolution molecules will not create a significant background fluorescencethat could interfere with the measurement of surface fluorescence. Insome embodiments, the member of the FRET pair attached to the ampliconis fluorescent. In some embodiments, the member of the FRET pair that isattached to the amplicon has an emission spectrum that is different thanthe member of the FRET pair that is attached to the surface. When theFRET pairs interact upon binding of the amplicon to the probe, in somecases, the interaction will result in a shift in the fluorescenceemission peak, for example due to charge transfer between the members ofthe FRET pair.

The methods and systems of the present invention comprise real-timemicroarray systems. Some of the advantages of RT-microarray systems overconventional microarray platforms are in their higher detection dynamicrange, lower minimum detection level (MDL), robustness, fastermeasurement times, and lower sensitivity to array fabrication systematicerrors, analyte binding fluctuation, and biochemical noise, as well asin the avoidance of the washing step required for conventionalmicroarrays.

One aspect of the invention is a method of measuring binding of analytesto a plurality of probes on surface in “real time”. As used herein inreference to monitoring, measurements, or observations of binding ofprobes and analytes of this invention, the term “real-time” refers tomeasuring the status of a binding reaction while that reaction isoccurring, either in the transient phase or in biochemical equilibrium.Real time measurements are performed contemporaneously with themonitored, measured, or observed binding events, as opposed tomeasurements taken after a reaction is fixed. Thus, a “real time” assayor measurement generally contains not only the measured and quantitatedresult, such as fluorescence, but expresses this at various time points,that is, in hours, minutes, seconds, milliseconds, nanoseconds, etc.“Real time” includes detection of the kinetic production of signal,comprising taking a plurality of readings in order to characterize thesignal over a period of time. For example, a real time measurement cancomprise the determination of the rate of increase or decrease in theamount of analyte bound to probe. While the measurement of signal inreal-time can be useful for determining rate by measuring a change inthe signal, in some cases the measurement of no change in signal canalso be useful. For example, the lack of change of a signal over timecan be an indication that hybridization has reached steady-state.

The measurements are performed in real time with a plurality of probesand amplicons. The invention is useful for measuring probes andamplicons that bind specifically to one another. A probe and an ampliconpair that specifically bind to one another can be a specific bindingpair.

One aspect of the invention is a method of measuring binding betweenanalyte and probe which lowers, and in some cases eliminates the noisewhich is present in conventional microarrays and which decreases thequality of the amplicon-probe binding information. In conventionalmicroarrays and most of the affinity-based biosensors, the detection andincubation phases of the assay procedure are carried out at differenttimes. First the hybridization is carried out in the presence of thefluid, then the fluid is removed, the array is rinsed and dried, andconventional detection via scanning and/or imaging technique used toassess the captured targets in the dry phase. This type of conventionalmicroarray technique is not amenable to the multiplex Q-PCR reactions ofthe present invention.

The following analysis illustrates inherent problems with conventionalmicroarray analysis techniques, and the advantages of the real timemicroarray systems of the present invention in improving the quality ofthe binding measurement. Let x(t) denote the total number of capturedanalyte in a given spot of the microarray and/or affinity-basedbiosensor at a given time instant t. Furthermore, let x(t) denote theexpected value of x(t) when the incubation process has reachedbiochemical equilibrium. A typical microarray procedure is focused onestimating x(t), and using its value as an indication of the analyteconcentration in the sample; in fact, most data analysis techniquesdeduce their results based on x(t). Nevertheless, if we measure thenumber of captured analytes at time t₁ in the equilibrium, for any givenmicroarray spot it holds that x(t₁)≠ x(t). This is due to the inherentbiochemical noise and other uncertainties of the system. This phenomenonis illustrated in FIG. 6, where the number of captured analytes in eachspot of the microarray fluctuates in time, even in biochemicalequilibrium. Accordingly, a single measurement taken at time t₁, whichis what conventional microarray experiments provide, essentially samplesa single point of the random process of analyte binding.

Now, consider the case where we are able measure x(t) multiple times, inreal-time without the necessity of stopping the incubation and analytebinding reaction. This platform, which we call the real-time microarrays(RT-μArrays), has many advantages over the conventional method. In someembodiments of RT-μArrays, the kinetic of the bindings can be observed.Therefore, one can test whether the system has reached biochemicalequilibrium or not. In other embodiments, multiple samples of x(t) aremeasured (see FIG. 7), and different averaging techniques and/orestimation algorithms can be used to estimate x(t) and othercharacteristics of process x(t).

FIG. 8 shows a block diagram of the errors associated with conventionalDNA microarrays. These may be categorized into three stages:pre-hybridization (steps 1 and 2), hybridization (step 3), andpost-hybridization (steps 4 and 5). The pre-hybridization errors arisefrom sample purification variations and the errors or variations inreverse transcribing mRNA to cDNA, in generating in vitro transcribedRNA complementary to the cDNA (cRNA, or IVT RNA), and or in labeling theanalytes (step 1), and the errors arising from non-uniform probespotting and or synthesis on the array (step 2). The hybridizationerrors arise from the inherent biochemical noise, cross-hybridization tosimilar targets, and the probe saturation (step 3). Post-hybridizationerrors include washing artifacts, image acquisition errors (step 4), andsuboptimal detection (step 5). The most critical of these are probedensity variations (step 2), probe saturation and cross-hybridization(step 3) and washing artifacts (step 4).

The methods and systems of the present invention can compensate for allthe above errors except for those of sample preparation (step 1). Probedensity variations can be measured prior to incubation and thereforeaccounted for in post-processing (step 5), incubation noise can bereduced by taking many samples (rather than a single one), as mentionedearlier, probe saturation can be avoided by estimating targetconcentrations from the reaction rates, and finally washing is avoidedaltogether.

Methods

One aspect of the invention is a method comprising; (a) performing anucleic acid amplification on two or more nucleotide sequences toproduce two or more amplicons in a fluid in contact with the arraywherein the array comprises a solid surface with a plurality of nucleicacid probes at independently addressable locations; and (b) measuringthe hybridization of the amplicons to the two or more nucleic acidprobes while the fluid is in contact with the array to obtain anamplicon hybridization measurement.

In one embodiment the method involves the use of probe arrays in whicheach addressable location comprises a fluorescent moiety capable ofemitting a signal that is quenchable upon binding of an amplicon. Forexample, the quenchable moiety (e.g., a fluorescent moiety) is attachedto the probe on the array or in close physical proximity thereto. Thesurface of such array will emit signal from each addressable locationwhich can be detected using, for example, a series of lenses and a lightdetector (e.g., a CCD camera or CMOS image sensor). The primers and/orthe amplicons in the sample are tagged with a quencher moiety that canquench the signal from the quenchable moiety. When the quencher doesnot, itself, emit a light signal, there is little interference with thesignal from the array. This diminishes the noise in the measurement offluorescence from the array surface. During the course of a bindingreaction between amplicons and substrate-bound probes, the signal ateach addressable location is quenched. The signal at each addressablelocation is measured in real time, for example, by a CCD camera focusedon the array surface. As the signal at any location changes as a resultof binding and quenching, the change is measured. These measurementsover time allow determination of the kinetics of the reaction which, inturn, allows determination of the concentration of amplicons in thesample.

Alternatively if the primers and/or amplicons can be labeled with alight-emitting reporter, such as a fluorescent label, and the backgroundsignal from these species can be diminished by focusing the detector atthe array surface, thereby enhancing the signal from the surface boundmoieties, and minimizing the noise from signal in solution. In addition,the interference from background fluorescence can be improved by havingfluorescent labels on the surface with different emission spectra thanthe fluorescent moieties in solution on the primers and/or amplicons.

In another embodiment, the probes are attached to the surface of anarray comprising sensors, such as a CMOS sensor array, which produceelectrical signals that change as a result of binding events on theprobes. This also affords real time measurement of a plurality ofsignals on an array (Hassibi and Lee, IEEE Sensors journal, 6-6, pp.1380-1388, 2006, and Hassibi, A. “Integrated Microarrays,” Ph.D. ThesisStanford University, 2005.)

Accordingly, the methods of this invention allow real time measurementsof a plurality of binding events of amplicons to an array of probes on asolid support.

Probe and Amplicon, and Nucleotide Sequence

One aspect of the invention is the determination of the presence oramount of a nucleotide sequence. The term “nucleotide sequence” or“nucleic acid sequence” as used in this context refers to a sequence ofnucleotides of which it is desired to know the presence or amount. Thenucleotide sequence is typically RNA or DNA, or a sequence derived fromRNA or DNA. Examples of nucleotide sequences are sequences correspondingto natural or synthetic RNA or DNA including genomic DNA and messengerRNA. The length of the sequence can be any length that can be amplifiedinto amplicons, for example up to about 20, 50, 100, 200, 300, 400, 500,600, 700, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000 or more than10,000 nucleotides in length. It will be understood by those of skill inthe art that as the number of nucleotides gets very large, theamplification can be less effective.

An “amplicon” as used herein is a molecular species that is created fromthe amplification of a nucleotide sequence. An amplicon is typically apolynucleotide such as RNA or DNA or mixtures thereof, in which thesequence of nucleotides in the amplicon correlates with the sequence ofthe nucleotide sequence from which it was generated (i.e. eithercorresponding to or complimentary to the sequence). The amplicon can beeither single stranded or double stranded. In some embodiments, theamplicon is created using one or more primers that is incorporated intothe amplicon. In some embodiments, the amplicon is generated in apolymerase chain reaction or PCR amplification, wherein two primers areused, creating what can be referred to as either a pair of complementarysingle stranded amplicons or a double-stranded amplicon.

The terms “probe” as used herein refers to a molecular species thatbinds to an amplicon in solution. A single probe or a single amplicon isgenerally one chemical species. That is, a single amplicon or probe maycomprise many individual molecules. In some cases, a probe or ampliconmay be a set of molecules that are substantially identical. A “probe”can be any type of molecule that can specifically bind to an amplicon inorder to measure its amount in the fluid. A probe is a molecule that canspecifically hybridize to amplicons in solution.

The probes of the present invention are bound to the substrate or solidsurface. For instance, the probe can comprise biological materialsdeposited so as to create spotted arrays; and materials synthesized,deposited, or positioned to form arrays according to other technologies.Thus, microarrays formed in accordance with any of these technologiesmay be referred to generally and collectively hereafter for convenienceas “probe arrays.” Moreover, the term “probe” is not limited to probesimmobilized in array format. Rather, the functions and methods describedherein may also be employed with respect to other parallel assaydevices. For example, these functions and methods may be applied withrespect to probe-set identifiers that identify probes immobilized on orin beads, optical fibers, or other substrates or media. The constructionof various probe arrays of the invention are described in more detailbelow.

In some embodiments, the probe, the amplicon, and/or the nucleotidesequence comprise a polynucleotide. The terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule” as usedherein include a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). This term refersonly to the primary structure of the molecule. Thus, the term includestriple-, double- and single-stranded DNA, as well as triple-, double-and single-stranded RNA. It also includes modifications, such as bymethylation and/or by capping, and unmodified forms of thepolynucleotide. More particularly, the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and other polymers containing nonnucleotidic backbones. A nucleicacid of the present invention will generally contain phosphodiesterbonds, i.e. a natural nucleic acid, although in some cases, as outlinedbelow, nucleic acid analogs are included that may have alternatebackbones, comprising, for example, phosphoramide (Beaucage et al.,Tetrahedron 49(10):1925 (1993) and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al., J. Am. Chem. Soc. 11 1:2321 (1989),O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid (PNA) backbones and linkages (see Carlsson et al., Nature380:207 (1996)). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). These modifications of theribose-phosphate backbone may be done to facilitate the addition oflabels, or to increase the stability and half-life of such molecules inphysiological environments.

In some embodiments of the invention, oligonucleotides are used. An“oligonucleotide” as used herein is a single-stranded nucleic acidranging in length from 2 to about 1000 nucleotides, more typically from2 to about 500 nucleotides in length. In some embodiments, it is about10 to about 100 nucleotides, and in some embodiments, about 20 to about50 nucleotides. It can be advantageous to use an oligonucleotide inthese size ranges as probes.

In some embodiments of the invention, for example, expression analysis,the invention is directed toward measuring the nucleic acid ornucleotide sequence concentration in a sample. In some cases the nucleicacid concentration, or differences in nucleic acid concentration betweendifferent samples, reflects transcription levels or differences intranscription levels of a gene or genes. In these cases it can bedesirable to provide a nucleic acid sample comprising mRNA transcript(s)of the gene or genes, or nucleic acids derived from the mRNAtranscript(s). As used herein, a nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed fromthat cDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. Thus, suitable samplesinclude, but are not limited to, mRNA transcripts of the gene or genes,cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA,DNA amplified from the genes, RNA transcribed from amplified DNA, andthe like. All of the above can comprise the nucleotide sequence forwhich the presence or amount is measured.

In the simplest embodiment, such a nucleic acid sample is the total mRNAor a total cDNA isolated and/or otherwise derived from a biologicalsample. The term “biological sample”, as used herein, refers to a sampleobtained from an organism or from components (e.g., cells) of anorganism. The sample may be of any biological tissue or fluid.Frequently the sample will be a “clinical sample” which is a samplederived from a patient. Such samples include, but are not limited to,sputum, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes.

The nucleic acid (either genomic DNA or mRNA) may be isolated from thesample according to any of a number of methods well known to those ofskill in the art. One of skill will appreciate that where alterations inthe copy number of a gene are to be detected genomic DNA is preferablyisolated. Conversely, where expression levels of a gene or genes are tobe detected, preferably RNA (mRNA) is isolated.

Methods of isolating total mRNA are well known to those of skill in theart. For example, methods of isolation and purification of nucleic acidsare described in detail in Chapter 3 of Laboratory Techniques inBiochemistry and Molecular Biology Hybridization With Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed.Elsevier, N.Y. (1993) and Chapter 3 of Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed.Elsevier, N.Y. (1993)).

In some embodiments, the probe may comprise a polypeptide. Polypeptidesand proteins can have specific binding properties. For instance, anenzyme can have a region that binds specifically with a substrate suchas an amplicon. Antibodies, which can have very specific bindingproperties are polypeptides that can be used as probes.

Probes on a Solid Substrate

For the methods of the present invention, the probes are attached to asolid substrate. The solid substrate may be biological, nonbiological,organic, inorganic, or a combination of any of these, existing asparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides,semiconductor integrated chips etc. The solid substrate is preferablyflat but may take on alternative surface configurations. For example,the solid substrate may contain raised or depressed regions on whichsynthesis or deposition takes place. In some embodiments, the solidsubstrate will be chosen to provide appropriate light-absorbingcharacteristics. For example, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂SiN₄, modified silicon, or any one of a variety of gels or polymers suchas (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, or combinations thereof. The solid support and thechemistry used to attach the solid support are described in detailbelow.

The substrate can be a homogeneous solid and/or unmoving mass muchlarger than the capturing probe where the capturing probes are confinedand/or immobilized within a certain distance of it. The mass of thesubstrate is generally at least 100 times larger than capturing'probesmass. In certain embodiments, the surface of the substrate is planarwith roughness of 0.1 nm to 100 nm, but typically between 1 nm to 10 nm.In other embodiments the substrate can be a porous surface withroughness of larger than 100 nm. In other embodiments, the surface ofthe substrate can be non-planar. Examples of non-planar substrates arespherical magnetic beads, spherical glass beads, and solid metal and/orsemiconductor and/or dielectric particles.

For the methods of the present invention, the plurality of probes may belocated in one addressable region and/or in multiple addressable regionson the solid substrate. In some embodiments the solid substrate hasabout 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500, 500-1,000,1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000,100,000-500,000, 500,000-1,000,000 or over 1,000,000 addressable regionswith probes.

In some embodiments it is also useful to have addressable regions whichdo not contain probe, for example, to act as control spots in order toincrease the quality of the measurement, for example, by using bindingto the spot to estimate and correct for non-specific binding.

Amplicon/Probe Hybridization or Binding

The methods of the present invention utilize the measurement of thehybridization or binding characteristics of multiple amplicons tomultiple probes in real-time. The method is particularly useful forcharacterizing the binding of probes and amplicons which specificallybind or hybridize to one another. As used herein, a probe “specificallybinds” to a specific analyte if it binds to that analyte with greateraffinity than it binds to other substances in the sample.

The binding between the probes and the amplicons in the presentinvention occurs in solution; usually in an aqueous solution. An aqueoussolution is a solution comprising solvent and solute where the solventis comprised mostly or completely of water. The methods of theinvention, however, can be used in any type of solution where thebinding between a probe and an amplicon can occur and be observed.

Typically, the probe and amplicon will specifically bond byhybridization, but in some cases the binding can be through othermolecular recognition mechanisms. Molecular recognition generallyinvolves detecting binding events between molecules. The strength ofbinding can be referred to as “affinity”. Affinities between biologicalmolecules are influenced by non-covalent intermolecular interactionsincluding, for example, hydrogen bonding, hydrophobic interactions,electrostatic interactions and Van der Waals forces. In multiplexedbinding experiments, such as those contemplated here, a plurality ofanalytes and probes are involved. For example, the experiment mayinvolve testing the binding between a plurality of different nucleicacid molecules or between different proteins. In such experimentsanalytes preferentially will bind to probes for which they have thegreater affinity. Thus, determining that a particular probe is involvedin a binding event indicates the presence of an amplicon in the samplethat has sufficient affinity for the probe to meet the threshold levelof detection of the detection system being used. One may be able todetermine the identity of the binding partner based on the specificityand strength of binding between the probe and amplicon.

The specific binding can be, for example, a receptor-ligand,enzyme-substrate, antibody-antigen, or a hybridization interaction. Theprobe/amplicon binding pair can be nucleic acid to nucleic acid, e.g.DNA/DNA, DNA/RNA, RNA/DNA, RNA/RNA, RNA. The probe/amplicon binding paircan be a polypeptide and a nucleic acid, e.g. polypeptide/DNA andpolypeptide/RNA, such as a sequence specific DNA binding protein. Theprobe/amplicon binding pair or amplicon/probe binding pair can be anynucleic acid and synthetic DNA/RNA binding ligands (such as polyamides)capable of sequence-specific DNA or RNA recognition. The probe/ampliconbinding pair can comprise natural binding compounds such as naturalenzymes and antibodies, and synthetic binding compounds. Theprobe/amplicon binding can comprise aptamers, which are nucleic acid orpolypeptide species that have been engineered to have specific bindingproperties, usually through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment).

The hybridization of the amplicon to the probe results in a change insignal. Generally, the signal due to hybridization will be proportionalto the amount of hybridized amplicon. While it can be advantageous tohave the proportionality be relatively strict (e.g., a two fold changeof the amplicon amount and a two fold change in hybridization signal),one of skill will appreciate that the proportionality can be morerelaxed and even non-linear. Thus, for example, an assay where a 5 folddifference in concentration of the amplicon results in a 3 to 6 folddifference in hybridization intensity is sufficient for most purposes.Where more precise quantification is required appropriate controls canbe run to correct for variations introduced in sample preparation andhybridization as described herein. Where simple detection of thepresence or absence of a nucleotide sequence or large differences ofchanges in nucleic acid concentration are desired, no elaborate controlor calibration is required.

Nucleic Acid Systems

One particularly useful aspect of the present invention involvesspecific hybridization between an amplicon and a probe, where bothcomprise nucleic acids.

As nucleic acid probe is a nucleic acid capable of binding to a targetnucleic acid of complementary sequence through one or more types ofchemical bonds, usually through complementary base pairing, usuallythrough hydrogen bond formation. The nucleic acid probe may includenatural (i.e. A, G, C, or T) or modified bases (7-deazaguanosine,inosine, etc.). In addition, the bases in nucleic acid probe may bejoined by a linkage other than a phosphodiester bond, so long as it doesnot interfere with hybridization. Thus, nucleic acid probes may bepeptide nucleic acids in which the constituent bases are joined bypeptide bonds rather than phosphodiester linkages. The nucleic acidprobes can also comprise locked nucleic acids (LNA), LNA, often referredto as inaccessible RNA, is a modified RNA nucleotide. The ribose moietyof the LNA nucleotide is modified with an extra bridge connecting 2′ and4′ carbons. The bridge “locks” the ribose in 3′-endo structuralconformation, which is often found in A-form of DNA or RNA. LNAnucleotides can be mixed with DNA or RNA bases in the oligonucleotide.Such oligomers are commercially available. The locked riboseconformation can enhance base stacking and backbone pre-organization,and can increase the thermal stability (melting temperature) ofoligonucleotides.

In the present invention, the specific hybridization of anoligonucleotide probe to the target nucleic acid can be measured inreal-time. An oligonucleotide probe will generally hybridize, bind, orduplex, with a particular nucleotide sequence of an amplicon understringent conditions even when that sequence is present in a complexmixture. The term “stringent conditions” refers to conditions underwhich a probe will hybridize preferentially to its target subsequence,and to a lesser extent to, or not at all to, other sequences.

For nucleic acid systems, the nucleic acid probes of the presentinvention are designed to be complementary to a nucleic acid targetsequence in an amplicon, such that hybridization of the amplicon and theprobes of the present invention occurs. This complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, an oligonucleotide probe that isnot substantially complementary to a nucleic acid analyte will nothybridize to it under normal reaction conditions.

The methods of the present invention thus can be used, for example, todetermine the sequence identity of a nucleic acid amplicon in solutionby measuring the binding of the amplicon with known probes. The sequenceidentity can be determined by comparing two optimally aligned sequencesor subsequences over a comparison window or span, wherein the portion ofthe polynucleotide sequence in the comparison window may optionallycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical subunit (e.g.nucleic acid base or amino acid residue) occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The methods of the current invention when applied to nucleic acids, canbe used for a variety of applications including, but not limited to, (1)mRNA or gene expression profiling, involving the monitoring ofexpression levels for example, for thousands of genes simultaneously.These results are relevant to many areas of biology and medicine, suchas studying treatments, diseases, and developmental stages. For example,microarrays can be used to identify disease genes by comparing geneexpression in diseased and normal cells; (2) comparative genomichybridization (Array CGH), involving the assessment of large genomicrearrangements; (3) SNP detection arrays for identifying for SingleNucleotide Polymorphisms (SNP's) in the genome of populations; andchromatin immunoprecipitation (chIP) studies, which involve determiningprotein binding site occupancy throughout the genome, employingChIP-on-chip technology.

The present invention can be very sensitive to differences in bindingbetween amplicons comprising different nucleic acid species, in somecases, allowing for the discrimination down to a single base pairmismatch. And because the present invention allows the simultaneousmeasurement of multiple binding events, it is possible to analyzeseveral amplicons simultaneously, where each is intentionally mismatchedto different degrees. In order to do this, a “mismatch control” or“mismatch probe” which are probes whose sequence is deliberatelyselected not to be perfectly complementary to a particular targetsequence can be used, for example in expression arrays. For eachmismatch (MM) control in an array there, for example, exists acorresponding perfect match (PM) probe that is perfectly complementaryto the same particular target sequence of an amplicon. In “generic”(e.g., random, arbitrary, haphazard; etc.) arrays, since the targetnucleic acid(s) are unknown, perfect match and mismatch probes cannot bea priori determined, designed, or selected. In this instance, the probescan be provided as pairs where each pair of probes differs in one ormore pre-selected nucleotides. Thus, while it is not known a prioriwhich of the probes in the pair is the perfect match, it is known thatwhen one probe specifically hybridizes to a particular target sequenceof an amplicon, the other probe of the pair will act as a mismatchcontrol for that target sequence. It will be appreciated that theperfect match and mismatch probes need not be provided as pairs, but maybe provided as larger collections (e.g., 3, 4, 5, or more) of probesthat differ from each other in particular preselected nucleotides. Whilethe mismatch(s) may be located anywhere in the mismatch probe, terminalmismatches are less desirable as a terminal mismatch is less likely toprevent hybridization of the target sequence. In a particularlypreferred embodiment, the mismatch is located at or near the center ofthe probe such that the mismatch is most likely to destabilize theduplex with the target sequence under the test hybridization conditions.In a particularly preferred embodiment, perfect matches differ frommismatch controls in a single centrally-located nucleotide.

It will be understood by one of skill in the art that control of thecharacteristics of the solution such as the stringency are important inusing the present invention to measure the binding of a amplicon-probepair, or the concentration of an amplicon. A variety of hybridizationconditions may be used in the present invention, including high,moderate and low stringency conditions; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al, hereby incorporatedby reference. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, “Overview of principles of hybridization and the strategy ofnucleic acid assays” (1993). In some embodiments, highly stringentconditions are used. In other embodiments, less stringent hybridizationcondition; for example, moderate or low stringency conditions may beused, as known in the art; see Maniatis and Ausubel, supra, and Tijssen,supra. The hybridization conditions may also vary when a non-ionicbackbone, i.e. PNA is used, as is known in the art.

Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences tend to hybridize specificallyat higher temperatures. Generally, stringent conditions can be selectedto be about 5 degree. C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. (As the targetanalyte sequences are generally present in excess, at T_(m), 50% of theprobes are occupied at equilibrium). Typically, stringent conditionswill be those in which the salt concentration is at least about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides). Stringent conditions may, also be achieved with theaddition of destabilizing agents such as formamide.

Binding/Hybridization Kinetics

One aspect of the current invention is the use of the measurement of thebinding kinetics to characterize binding of multiple probes andamplicons in solution. The term “binding kinetics” or “hybridizationkinetics” as used herein refers to the rate at which the binding of theanalyte to the probe occurs in a binding/hybridization reaction. Theterm “binding reaction” as used herein describes the reaction betweenprobes and amplicons. The term “hybridization reaction” is a bindingreaction wherein the binding comprises hybridization, for example ofcomplementary nucleic acids. In some cases, binding reaction refers tothe concurrent binding reactions of multiple amplicons and probes, andin other cases, the term binding reaction refers to the reaction betweena single probe with a single amplicon. The meaning will be clear fromthe context of use. The kinetic measurements can be expressed as theamount of amplicon bound to the probe as a function of time. The bindingor hybridization kinetics can provide information about thecharacteristics of the probe-amplicon binding such as the strength ofbinding, the concentration of amplicon, the competitive binding of anamplicon, the density of the probes, or the existence and amount ofcross-hybridization.

In order to determine binding kinetics, the signal at multiple timepoints must be determined. The signal at least two time points isrequired. In most cases, more than two time points will be desired inorder to improve the quality of the kinetic information. In someembodiments the signal at, 2-10, 10-50, 50-100, 100-200, 200-400,400-800, 800-1600, 1600-3200, 3200-6400, 6400-13000, or higher than13,000 time points will be measured. One of ordinary skill in the artcan determine the effective number of points for a given embodiment. Forexample, where few points are obtained, the quality of information aboutthe binding event can be low, but where the number of data points isvery high, the data quality may be high, but the handling, storage, andmanipulation of the data can be cumbersome.

The frequency at which the signal is measured may depend on the kineticsof the binding reaction or reactions that are being monitored. As thefrequency of measurements gets lower, the time between measurements getslonger. One way to characterize a binding reaction is to refer to thetime at which half of the analyte will be bound (t_(1/2)). The bindingreactions of the invention can have a (t_(1/2)) from on the order ofmilliseconds to on the order of hours, thus the frequency ofmeasurements can vary by a wide range. The time between measurementswill generally not be evenly spaced over the time of the bindingreaction. In some embodiments, a short time between of measurements willbe made at the onset of the reaction, and the time between measurementswill be longer toward the end of the reaction. One advantage of thepresent invention is the ability to measure a wide range of bindingrates. A high initial frequency of measurements allows thecharacterization of fast binding reactions which may have higherbinding, and lower frequency of measurements allows the characterizationof slower binding reactions. For example, points can initially bemeasured at a time between points on the order of a microsecond, thenafter about a millisecond, points can be measured at a time betweenpoints on the order of a millisecond, then after about a second, timepoints can be measured at a time between points on the order of asecond. Any function can be used to ramp the change in measurementfrequency with time. In some cases, as described below, changes in thereaction conditions, such as stringency or temperature changes will bemade during a reaction, after which it may be desirable to change thefrequency of measurements to measure the rates of reaction which will bechanged by the change in reaction condition.

In some embodiments, a probe will have substantially no amplicon boundto it at the beginning of the binding reaction, then the probe will beexposed to a solution containing the analyte, and the analyte will beginto bind, with more analyte bound to the probe with time. In some cases,the reaction will reach saturation, the point at which all of theanalyte that is going to bind has bound. Generally, saturation willoccur when a reaction has reached steady state. At steady state, in agiven time period, just as many analytes are released as new analytesare bound (the on rate and off rate are equal). In some cases, with verystrong binding, where the off-rate for the analyte is essentially zero,saturation will occur when substantially all of the analyte that canbind to the probe will have bound, has bound. Thus, while it isadvantageous to measure a change in signal with time that can becorrelated with binding kinetics, the measurement of a signal that doesnot change with time also provides information in the context of areal-time experiment, and can also be useful in the present invention.For example, in some cases the absence of a change in the signal willindicate the level of saturation. In other cases the absence of a changein signal can indicate that the rate of the reaction is very slow withrespect to the change in time measured. It is thus a beneficial aspectof this invention to measure binding event in real time both wheresignals change with time and where the signals do not change with time.

One aspect of the methods of the present invention is the measurement ofconcentration of an amplicon from the measurement of binding kinetics.Since amplicon binding rate can be concentration-dependant, we canestimate the amplicon abundance in the sample solution using bindingrates.

In some embodiments, the concentration of an analyte such as an ampliconcan be determined by equations relating to the kinetics of thehybridization process. For example, suppose that the number of probes ata particular spot on the array prior to any hybridization is given byP₀. The probability of a specific target such as an amplicon binding tothe probe site is given by

Prob(binding)=kProb(target and probe in close proximity)Prob(probe isfree),  (1)

where k≦1 depends of the bonds between the probe and the target andessentially a function of temperature, incubation conditions, and probedensity. Here, the first probability is proportional to the number oftarget molecules available whereas the second probability is

Probe(probe is free)=P(t)P ₀,  (2)

where P(t) is the number of available probes at time t, i.e., those arenot yet bound to any target. If we thus denote the forward and backwardstarget/probe binding reaction rates by K₊ and K⁻, respectively, we maywrite the following differential equation for the available probeconcentration P(t):

$\begin{matrix}{\frac{{P(t)}}{t} = {{{- \frac{K_{+}}{P_{0}}}{P(t)}\left( {C - P_{0} - {P(t)}} \right)} + {K_{-}\left( {P_{0} - {P(t)}} \right)}}} & (3)\end{matrix}$

where C is the original target quantity in the solution so thatC−(P₀−P(t)) represents the available target density at time t. The aboveis a Riccati differential equation that can be solved in closed form.However, instead of doing so, we can note that for small values oft wehave P(t)≅P₀, so that the differential equation becomes

$\begin{matrix}{\frac{{P(t)}}{t} = {{- \frac{K_{+}}{P_{0}}}{P(t)}{C.}}} & (4)\end{matrix}$

This a first-order linear differential equation with time constantτ=P₀/K₊C. Accordingly, the target density can be determined from thereaction rate (or time constant) of P(t). In other words, using manysample measurements of P(t) at different times and fitting them to thecurve

$\begin{matrix}{{P(t)} = {P_{0}{\exp \left( {{- \frac{K_{+}}{P_{0}}}C} \right)}t}} & (5)\end{matrix}$

allows us to estimate the target quantity C. In this case, the reactionrate (or inverse of the time constant) is proportional to the targetconcentration and inversely proportional to the probe density, somethingthat has been observed in experiments.

One can also attempt to estimate C from the steady-state value of P(t),i.e., P_(∞). This can be found by setting dP(t)/dt=0 in the originalRiccati equation which leads to a quadratic equation for P_(∞). In somesimple cases, the solution to this quadratic equation can beconsiderably simplified.

When the target concentration is low: In this case, we can assume P₀>>C,so that we obtain

P _(∞) =P ₀ −C,  (6)

i.e., the reduction in available probes is equal to the targetconcentration.

When the target concentration is high: In this case, we can assume thatP₀<<C. so that we obtain

$\begin{matrix}{P_{\infty} = {\frac{K_{-}}{K_{+}} \cdot {\frac{P_{0}^{2}}{C}.}}} & (7)\end{matrix}$

In this case, the number of remaining probes is inversely proportionalto the target concentration. This corresponds to probe saturation, whichgenerally is not as good a method of determining C as determining Cbased on the reaction rate near the beginning of the reaction.

One aspect of the present invention is the determination of the bindingor hybridization of amplicon to probe by measuring the rate near thebeginning of the reaction. In addition to providing a more reliableestimate of C, measurement near the beginning of the reaction canshorten the time that is required to measure analyte binding over thetime required for measuring binding from saturation. In some embodimentsof the invention, the binding is measured during the time for less thanabout the first 0.1, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 18, 20, 25,30, 40, 50, 60, 70, 80, or 90 percent of the amplicon to bind ascompared to the amount of analyte bound at saturation. In someembodiments, the binding kinetics are determined in a time for less thanabout the first 20% of the amplicon to bind. In some embodiments, thebinding kinetics are determined in a time for less than about the first1-2% of the amplicon to bind.

Changing Conditions During the Binding Experiment

One aspect of the methods of the present invention is a step of changingthe conditions during the binding experiment. In conventionalmicroarrays where only the end-point is determined, only a single set ofbinding conditions can be tested. In the methods of the presentinvention, the binding conditions can be changed in order to exploremultiple sets of binding conditions during the same binding experiment.The condition which is changed can be, for example, any condition thataffects the rate of binding of analyte to probe. The condition which ischanged can be, for example, temperature, pH, stringency, analyteconcentration, ionic strength, an electric field, or the addition of acompetitive binding compound.

In some embodiments, the condition that is changed changes the rate ofbinding or hybridization. When measuring the binding of multipleamplicons to probes in the same binding medium, as in the presentinvention, the kinetics of binding can vary widely for differentamplicon-probe combinations. The binding rate conditions can be varied,for example, by changing the temperature, concentration, ionic strength,pH, or by applying an electric potential. The binding rates for thedifferent amplicons can in some cases vary by many orders of magnitude,making it difficult and time consuming to measure the binding of all theanalytes in one binding experiment. This ability to change the rateconditions can be used to improve the measurement of binding formultiple amplicons that bind at different rates, for example byperforming the initial part of the experiment under slower rateconditions, such that rapidly binding amplicons can be readily measured,then raising the rate conditions such that more slowly binding ampliconscan be readily measured. This method of changing the binding rate duringthe binding experiment can also be used for better characterization of asingle amplicons or single set of amplicons in solution, for instance,using binding rate conditions to measure the initial portion of thebinding kinetics, then increasing the binding rate conditions to measurethe later portion of the binding kinetics for a single analyte, forexample, to establish the level of saturation. It will be understood bythose of skill in the art that this method of changing the rateconditions can result in both improved quality of measurements, such asthe measurement of amplicon concentration, and/or in a savings of time.With the present method, for example by measuring the kinetics ofbinding, then changing the conditions to increase the rate of binding ofweaker binding species, the time of the binding experiment can bereduced by greater than about 10%, 20%, 50%, 75%, or by a factor of 2,4, 8, 10, 50, 100, 1000 or greater than 1000 over the times needed toobtain the same quality information using end-point binding methods.

In some embodiments, the condition that is changed is the stringency. Asdescribed above, the stringency can be changed by many factors includingtemperature, ionic strength, and the addition of compounds such asformamide. In some embodiments of the present invention, the medium isat one stringency at the beginning of the binding reaction, and at alater point the stringency of the medium is changed. This method can beused where different analytes or sets of analytes have differenthybridization characteristics, for example, allowing the measurement ofthe binding of one set of amplicons with a high stringency, thenallowing the measurement of another set of amplicons at a lowerstringency in the same medium as part of the same binding experiment.This method can also be used for the characterization of binding for asingle amplicon by, for instance, measuring binding at high stringencyat an initial portion of the binding reaction, then lowering thestringency and measuring a later portion of the binding reaction. Theability to change stringency can also be used to create conditions wherea bound amplicon becomes unbound, allowing, for instance, themeasurement of the kinetics of binding at one stringency, followed bythe measurement of release of the analyte into solution upon raising thestringency. This method also allows the binding of an amplicon to aprobe to be measured multiple times, for example, by measuring thekinetics of binding of the amplicon under one set of stringencyconditions, changing the stringency to release the amplicon, forinstance, by raising the stringency, then measuring the kinetics ofbinding of the amplicon a subsequent time by changing the stringencyconditions again, for example by lowering the stringency. Thus theability to change the stringency during the binding reaction allows forthe measurement of any number of binding and unbinding reactions withthe same set of probes and amplicons.

In some embodiments of the method of the present invention, an electricpotential is applied during the binding reaction to the fluid volume toelectrically change the stringency of the medium. In some embodiments,the system will provide an electrical stimulus to the capturing regionusing an electrode structure which is placed in proximity of thecapturing region. If the amplicon is an electro-active species and/orion, the electrical stimulus can apply an electrostatic force of theanalyte. In some embodiments the electrical potential is direct current(DC). In some embodiments, the electric potential is time-varying. Insome embodiments the electric potential has both DC and time varyingaspects. Their amplitude of the applied potential can be between 1 mV to10V, but typically between 10 mV to 100 mV. The frequency oftime-varying signal is between 1 Hz to 1000 MHz, but typically between100 Hz to 100 kHz.

Detection of Signals

For the methods of the present invention, a signal is detected that canbe correlated with the binding of amplicons to the plurality of probes.The type of signals appropriate for the invention is any signal that canbe amount of amplicon bound to the plurality of probes. Appropriatesignals include, for example, electrical, electrochemical, magnetic,mechanical, acoustic, or electromagnetic (light) signals. Examples ofelectrical signals useful in the present invention that can becorrelated with amplicon binding are capacitance and/or impedance. Forexample, amplicons labeled with metals or metal clusters can change thecapacitance and/or the impedance of a surface in contact with a fluid,allowing the amount of analyte bound to the probe on the surface to bedetermined. The electrical measurement can be made at any frequencyincluding DC, 0-10 Hz, 10-100 Hz, 100-1000 Hz, 1 KHz-10 KHz, 10 KHz-100KHz, 100 KHz-1 MHz, 1 MHz-10 MHz, 10 MHz-100 MHz, 100 MHz-1 GHz, orabove 1 &Hz. In some embodiments, impedance spectroscopy can be usedwhich obtains impedance versus frequency for any range of frequencieswithin the range of frequencies described above. Examples ofelectrochemical signals useful in the present invention that can becorrelated with amplicon binding include amperometric and voltammetricmeasurements, and/or measurements that involve the oxidation orreduction of redox species. For example, the amplicon can be labeledwith a compound which undergoes an oxidation or reduction reaction at aknown redox potential, and the oxidative or reductive current can becorrelated with the amount of amplicon bound to surface probes. Examplesof mechanical signals include the use of microelectromechanical (MEMS)devices. For example, the binding of amplicon to probe on the surface ofa small surface feature, such as a cantilever, can change the mass ofthe surface feature, the vibration frequency of which can then becorrelated with the amount of analyte bound to the probe. Generally, thehigher the mass, the lower the vibration frequency. Examples of acousticsignals include surface acoustic wave (SAW), and surface plasmonresonance signals. A surface acoustic wave (SAW) is an acoustic wavetraveling along the surface of a material having some elasticity, withamplitude that typically decays exponentially with the depth of thesubstrate. The binding of labeled or unlabeled amplicon to probe on asurface can change the SAW characteristics, e.g. amplitude, frequency ina manner that can be correlated with the amount of analyte bound to aprobe. Surface plasmon resonance relies on surface plasmons, also knownas surface plasmon polaritons, which are surface electromagnetic wavesthat propagate parallel, usually along a metal/dielectric interface.Since the wave is on the boundary of the metal and the external medium(water for example), these oscillations are very sensitive to any changeof this boundary, such as the adsorption of molecules to the metalsurface. The binding of labeled or unlabeled analyte to a probe attachedto the surface can change the frequency of the resonant surface plasmonin a manner that can be correlated with the amount of amplicon bound tothe probes.

Particularly useful signals for the methods of the present invention areelectromagnetic (light) signals. Examples of optical signals useful inthe present invention are signals from fluorescence, luminescence, andabsorption. As used herein, the terms “optical”, “electromagnetic” or“electromagnetic wave” and “light” are used interchangeably.Electromagnetic waves of any frequency and wavelength that can becorrelated to the amount of analyte bound to probe on the surface can beused in the present invention including gamma rays, x-rays, ultravioletradiation, visible radiation, infrared radiation, and microwaves. Whilesome embodiments are described with reference to visible (optical)light, the descriptions are not meant to limit the embodiments to thoseparticular electromagnetic frequencies.

For the methods of the present invention it is desired that the signalchanges upon the binding of the analyte to the probe in a manner thatcorrelates with the amount of amplicon bound. In some cases, the changein signal will be a change in intensity of the signal. In someembodiments, the signal intensity will increase as more amplicon isbound to probe. In some embodiments, the signal intensity will decreaseas more amplicon is bound to probe. In some embodiments, the change insignal is not a change in intensity, but can be any other change in thesignal that can be correlated with amplicon binding to probe. Forexample, the change in signal upon binding of the amplicon can be achange in the frequency of the signal. In some embodiments, the signalfrequency will increase as more amplicon is bound to probe. In someembodiments, the signal frequency will decrease as more amplicon isbound to the probe.

The signal that is measured is generally the signal from the region ofthe solid surface. In some embodiments signal from the solution is usedas the signal that can be correlated with the amount of amplicon boundto the probe. The measurement of hybridization of the amplicons to theprobes provides an amplicon hybridization measurement. The ampliconhybridization measurement can, in some embodiments, be correlated withthe amount of amplicon bound to the probe. As used herein, theconcentration of a substance such as the amplicon is the amount of thesubstance per volume of fluid in which the amplicon is dissolved. Thus,a measurement of the concentration of the amplicon will provide ameasurement of the amount of the amplicon when the volume of the fluidis known.

In some embodiments of the methods of the present invention, labels areattached to the amplicons and/or the probes. Any label can be used onthe amplicon or probe which can be useful in the correlation of signalwith the amount of analyte bound to the probe. It would be understood bythose of skill in the art that the type of label which is used on theamplicon and/or probe will depend on the type of signal which is beingused, for example, as described above, a dense label for a mechanicalsignal, or a redox active label for a voltammetric measurement.

In some embodiments, the signal that can be correlated to the amount ofamplicon bound to probe is due to the buildup of label at the surface asmore amplicon is bound to the probes on the surface. For example, wherethe amplicon has a fluorescent label, as more amplicon binds, theintensity of the fluorescent signal can increase in a manner that can becorrelated with the amount of amplicon bound to probe on the surface.

In some embodiments, the signal that can be correlated to the amount ofamplicon bound to probe is due to a change in the signal from label onthe surface upon binding of the amplicon to the probe. For example,where a fluorescent label is on the surface, and the amplicon and/orprimers are labeled with a compound capable of changing the fluorescentsignal of the surface fluorescent label upon binding of the ampliconwith the probes, the change in signal can be correlated with the amountof amplicon bound to probe. In some embodiments, the amplicon is labeledwith a quencher, and the decrease in intensity from the surfacefluorescent label due to quenching is correlated to the increased amountof amplicon bound to probe. In some embodiments, the amplicon and/orprimers are labeled with a fluorescent compound which can undergo energytransfer with the fluorescent label on the surface such that theincrease in fluorescence from the amplicon fluorescent label and/or thedecrease in fluorescence from the surface fluorescent label can becorrelated with the amount of amplicon bound to probe. In someembodiments the surface fluorescent label is bound directly, e.g.covalently to the probe. In some embodiments, the surface fluorescentlabel is bound to the surface, is not bound to the probe, but is insufficient proximity that the binding of the amplicon to the probeproduces a change in signal from the surface fluorescent label that canbe correlated with the amount of amplicon bound to probe.

In some embodiments, the amplicon is unlabeled, and the concentration ofthe amplicon is determined by competitive binding with another labeledspecies, which competes with the amplicon for biding to a probe. Forexample, where we have a solution with an amplicon, A, whoseconcentration we want to determine, and we have a competitive bindingspecies, B, whose binding characteristics with probe and whoseconcentration are known, then using the present invention, we can use,for example, an array of probes on a surface to determine theconcentration of A by determining the amount of competitive binding of Bto a probe. For example, the probe is attached to a surface that isfluorescently labeled, and B is labeled with a quencher such that thelevel of quenching of the surface fluorescence can be correlated withthe amount of B bound to the probe. The rate of binding of B to theprobe is measured in real time, and the concentration of A is determinedby knowing the characteristics of A as a competitive binder. In someembodiments, the amount of the competitive binding species does not needto be known beforehand.

Electromagnetic Signals—Optical Methods

The use of optical detection provides a variety of useful ways ofimplementing the methods of the present invention. Optical methodsinclude, without limitation, absorption, luminescence, and fluorescence.

Some embodiments of the invention involve measuring light absorption,for example by dyes. Dyes can absorb light within a given wavelengthrange allowing for the measurement of concentration of molecules thatcarry that dye. In the present invention, dyes can be used as labels, onthe amplicon and/or primers or on the probe. The amount of dye can becorrelated with the amount of amplicon bound to the surface in order todetermine binding kinetics. Dyes can be, for example, small organic ororganometallic compounds that can be, for example, covalently bound tothe amplicon and/or primer or probe. Dyes which absorb in theultraviolet, visible, infrared, and which absorb outside these rangescan be used in the present invention. Methods such as attenuated totalreflectance (ATR), for example for infrared, can be used to increase thesensitivity of the surface measurement.

Some embodiments of the invention involve measuring light generated byluminescence. Luminescence broadly includes chemiluminescence,bioluminescence, phosphorescence, and fluorescence. In some embodiments,chemiluminescence, wherein photons of light are created by a chemicalreaction such as oxidation, can be used.

Fluorescent Systems

A useful embodiment of the present invention involves the use offluorescence. As used herein, fluorescence refers to the process whereina molecule relaxes to its ground state from an electronically excitedstate by emission of a photon. As used herein, the term fluorescencealso encompasses phosphorescence. For fluorescence, a molecule ispromoted to an electronically excited state generally by the absorptionof ultraviolet, visible, or near infrared radiation. The excitedmolecule then decays back to the ground state, or to a lower-lyingexcited electronic state, by emission of light. An advantage offluorescence for the methods of the invention is its high sensitivity.Fluorimetry may achieve limits of detection several orders of magnitudelower than for absorption. Limits of detection of 10.sup.-10 M or lowerare possible for intensely fluorescent molecules; in favorable casesunder stringently controlled conditions, the ultimate limit of detection(a single molecule) may be reached.

A wide variety of fluorescent molecules can be utilized in the presentinvention including small molecules, fluorescent proteins and quantumdots. Useful fluorescent molecules (fluorophores) include, but are notlimited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA);5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-HydroxyTryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); AFPs—AutoFluorescent Protein—(QuantumBiotechnologies); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™;Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™;Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™;Alizarin Complexion; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S;AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC(Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G;Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine;ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine;BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH);Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide(Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3;Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589;Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676;Bodipy FI; Bodipy FL ATP; Bodipy FI-Ceramide; Bodipy R6G SE; Bodipy TMR;Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP;Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC;BTC-SN; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; CalciumGreen-1 Ca.sup.2+Dye; Calcium Green-2 Ca.sup.2+; Calcium Green-SNCa.sup.2+; Calcium Green-C18 Ca.sup.2.sup.+; Calcium Orange; CalcofluorWhite; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A;Chromomycin A; CL-NERF; CMFDA; Coumarin Phalloidin; C-phycocyanine; CPMMethylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8;Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl;Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansylfluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3′ DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP);Dichlorodihydrofluorescein Diacetate (DCFH); DiD—Lipophilic Tracer; DiD(DiIC18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DiIC18(3));Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH);DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF 97; Eosin; Erythrosin;Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1);Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA;Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC;Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate;Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; Fluor X;FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2;Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF;Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); Gloxalic Acid;Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold);Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium;Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf;JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751(RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1;Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso TrackerGreen; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-indo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; Maxilon BrilliantFlavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X;Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514;Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium lodid (PL); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613[PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A; S65C; S65L;S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G;Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein;SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua;SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; UranineB; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H;Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3, Sybr Green, Thiazole orange(interchelating dyes), or combinations thereof.

Some embodiments of the present invention include the Alexa Fluor dyeseries (from Molecular Probes/Invitrogen) which cover a broad spectrumand match the principal output wavelengths of common excitation sourcessuch as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546,555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750. Someembodiments of the present invention include the Cy Dye fluorophoreseries (GE Healthcare), also covering a wide spectrum such as Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7. Some embodiments of the present inventioninclude the Oyster dye fluorophores (Denovo Biolabels) such asOyster-500, -550, -556, 645, 650, 656. Some embodiments of the presentinvention include the DY-Labels series (Dyomics), for example, withmaxima of absorption that range from 418 nm (DY-415) to 844 nm (DY-831)such as DY-415, -495, -505, -547, -548, -549, -550, -554, -555, -556,-560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636, -647,-648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700,-701, -730, -731, -732, -734, -750, -751, -752, -776, -780, -781, -782,-831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL. Some embodimentsof the present invention include the ATTO fluorescent labels (ATTO-TECGmbH) such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590,594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740.Some embodiments of the present invention include CAL Fluor and Quasardyes (Biosearch Technologies) such as CAL Fluor Gold 540, CAL FluorOrange 560, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL FluorRed 635, Quasar 670. Some embodiments of the present invention includequantum dots such as the EviTags (Evident Technologies) or quantum dotsof the Qdot series (Invitrogen) such as the Qdot 525, Qdot565, Qdot585,Qdot605, Qdot655, Qdot705, Qdot 800. Some embodiments of the presentinvention include fluorescein, rhodamine, and/or phycoerythrin.

FRET and Quenching

In some embodiments of the invention, fluorescence resonance energytransfer is used to produce a signal that can be correlated with thebinding of the amplicon to the probe. FRET arises from the properties ofcertain fluorophores. In FRET, energy is passed non-radiatively over adistance of about 1-10 nanometers between a donor molecule, which is afluorophore, and an acceptor molecule. The donor absorbs a photon andtransfers this energy non-radiatively to the acceptor (Forster, 1949, Z.Naturforsch. A4: 321-327; Clegg, 1992, Methods Enzymol. 211: 353-388).When two fluorophores whose excitation and emission spectra overlap arein close proximity, excitation of one fluorophore will cause it to emitlight at wavelengths that are absorbed by and that stimulate the secondfluorophore, causing it in turn to fluoresce. The excited-state energyof the first (donor) fluorophore is transferred by a resonance induceddipole-dipole interaction to the neighboring second (acceptor)fluorophore. As a result, the excited state lifetime of the donormolecule is decreased and its fluorescence is quenched, while thefluorescence intensity of the acceptor molecule is enhanced anddepolarized. When the excited-state energy of the donor is transferredto a non-fluorophore acceptor, the fluorescence of the donor is quenchedwithout subsequent emission of fluorescence by the acceptor. In thiscase, the acceptor functions as a quencher.

Pairs of molecules that can engage in fluorescence resonance energytransfer (FRET) are termed FRET pairs. In order for energy transfer tooccur, the donor and acceptor molecules must typically be in closeproximity (up to 7 to 10 nanometers. The efficiency of energy transfercan falls off rapidly with the distance between the donor and acceptormolecules.

Molecules that can be used in FRET include the fluorophores describedabove, and includes fluorescein, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS). Whether a fluorophore is a donor or an acceptor is definedby its excitation and emission spectra, and the fluorophore with whichit is paired. For example, FAM is most efficiently excited by light witha wavelength of 488 nm, and emits light with a spectrum of 500 to 650nm, and an emission maximum of 525 nm. FAM is a suitable donorfluorophore for use with JOE, TAMRA, and ROX (all of which have theirexcitation maximum at 514 nm).

In some embodiments of the methods of the present invention, theacceptor of the FRET pair is used to quench the fluorescence of thedonor. In some cases, the acceptor has little to no fluorescence. TheFRET acceptors that are useful for quenching are referred to asquenchers. Quenchers useful in the methods of the present inventioninclude, without limitation, Black Hole Quencher Dyes (BiosearchTechnologies such as BHQ-0, BHQ-1, BHQ-2, BHQ-3, BHQ-10; QSY Dyefluorescent quenchers (from Molecular Probes/Invitrogen) such as QSY7,QSY9, QSY21, QSY35, and other quenchers such as Dabcyl and Dabsyl; Cy5Qand Cy7Q and Dark Cyanine dyes (GE Healthcare), which can be used, forexample, in conjunction with donor fluors such as Cy3B, Cy3, or Cy5;DY-Quenchers (Dyomics), such as DYQ-660 and DYQ-661; and ATTOfluorescent quenchers (ATTO-TEC GmbH), such as ATTO 540Q, 580Q, 612Q.

In some embodiments of the methods of the invention, both the ampliconsand the probes have labels that are members of a FRET pair, and thelabels are attached such that when an amplicon binds to a probe, FRETwill occur between the labels, resulting in a change in signal that canbe correlated with the binding of amplicon to probe in real-time. Thechange in signal can be the decrease in the intensity of the donorand/or the increase in the intensity of the acceptor. The FRET pair canbe chosen such that emission wavelength of the donor fluorophore is farenough from the emission wavelength of the acceptor fluorophore, thatthe signals can be independently measured. This allows the measurementof both the decrease in signal from the donor and the increase in signalfrom the acceptor at the same time, which can result in improvements inthe quality of the measurement of binding. In some cases, the probe willhave a label that is the donor of the donor-acceptor pair. In somecases, the amplicon will have a label that is the donor of the donoracceptor pair.

In some embodiments of the methods of the invention, the amplicon willhave a fluorescent label that is a member of a FRET pair, and the othermember of the FRET pair will be attached to the surface, wherein themember of the FRET pair attached to the surface is not covalently linkedto the probe. In some cases, the amplicon will have a label that is thedonor of the donor-acceptor pair. In some cases, the amplicon will havea label that is the acceptor of the donor acceptor pair. In someembodiments, the member of the FRET pair that is attached to the surfaceis attached to an oligonucleotide which is attached to the surface (asurface-bound label). The oligonucleotide that is labeled with the FRETpair can be a nucleotide sequence that does not have a sequenceanticipated to specifically bind to an amplicon. The use of asurface-bound label allows for the labeling of multiple areas of anarray without having to label each specific binding probe. This cansimplify the production of the array and reduce costs. We have foundthat even though the surface-bound FRET pairs are not covalently boundto the probe, they can be sensitive to the binding of the ampliconlabeled with the other member of the FRET pair in a manner that allowsthe change in signal to be correlated with the amount of amplicon boundto probe.

In some embodiments of the methods of the present invention, theamplicon is labeled with a quencher, and the probe is labeled with adonor fluorophore. The amplicon is labeled with the quencher such thatwhen amplicon binds with the probe, the fluorescence from thefluorescent label on the probe is quenched. Thus, the signal, measuredin real-time, can be correlated with the amount of binding of theamplicon and the probe, allowing for the measurement of the kinetics ofthe binding. In some embodiments of the methods of the presentinvention, the amplicon is labeled with a quencher, and the probe islabeled with a donor fluorophore, that is not covalently attached to it.The quencher is labeled such that when amplicon binds with the probe,the fluorescence from the fluorescent label on the probe is quenched.Thus, the signal, measured in real-time, can be correlated with theamount of binding of the amplicon and the probe, allowing for themeasurement of the kinetics of the binding.

In some embodiments of the methods of the present invention, theamplicon is labeled with a quencher, and the surface is labeled with adonor fluorophore wherein the donor fluorophore is not covalently linkedto the probe (e.g. with a surface bound fluorescent label). The quencheris labeled such that when amplicon binds with the probe, thefluorescence from the fluorescent label on the surface is quenched.Thus, the signal, measured in real-time, can be correlated with theamount of binding of the amplicon and the probe, allowing for themeasurement of the kinetics of the binding.

Where the probe is labeled with a fluorophore, one aspect of theinvention is the use of an image of the fluorescently labeled probe onthe surface obtained before binding has occurred in order to effectivelyestablish a baseline signal for the state where no binding of ampliconto probe has occurred. In conventional arrays, in which unlabeled probeis treated with labeled amplicon, and the signal is measured afterhybridization and washing, it can be difficult to know exactly how muchprobe is actually on the array in the region of interest. Thus,differences in array manufacture can affect the quality of the data. Inthe present invention, where the probe is labeled with fluorophore, theimage of the labeled probe on the surface provides a measurement of theamount of probe actually on the surface, increasing the quality andreliability of the binding measurement.

One exemplary embodiment of a real-time microarray useful for carryingout the method of the invention is shown in FIGS. 9 and 10. FIG. 9Bshows a top view of a 4 by 4 microarray that has 16 independentlyaddressable spots, each spot having bound DNA probes, wherein the probesare labeled with fluorescent label. FIG. 9C shows a close up view of oneof the spots illustrating the attached probe of sequence (A), each probehaving a fluorescent label. FIG. 9D shows the close up view of a secondspot with attached probes of sequence (B), each probe having afluorescent label. FIG. 9A shows a side view of the array, showing thatthe array is in contact with the hybridization solution. FIG. 9represents a time at which no amplicon is bound to probe on the array.

FIG. 10 illustrates the same array as in FIG. 9 after hybridization forsome time with amplicons having a quencher attached. FIGS. 10A and 10Bshows a side view and top view of the array, still in contact with thehybridization solution. The different spots on the array in FIG. 10Bhave different light intensities, indicating that there is a differentamount of binding of amplicon at each spot, and therefore a differentamount of fluorescence from the spots. FIG. 10C shows a close up viewillustrating that a small amount of amplicon (A) has specifically bound(hybridized) to probe (A) resulting in quenching of each molecule ofprobe to which analyte is bound. FIG. 10D illustrates that a largeramount of amplicon (B) has specifically bound (hybridized) to probe (B),resulting in a higher level of quenching than observed for spot (A). Thesignal from each of the spots on the array can be measured at varioustime points during the binding reaction between analytes and probes,while the solution containing the analyte is in contact with the solidsurface of the microarray, allowing a real-time measurement of theamount of analyte-probe binding, and allowing the measurement of bindingkinetics at each spot.

The methods of the invention can be used to measure the presence oramount of nucleotide sequences in samples at low levels. In someembodiments, the presence or amount of nucleic acid can be measuredwhere the nucleic acid is present in a sample at the level of nanomoles,picomoles, femtomoles. In some cases it can determine the presence oramount of nucleotide sequences down to a single molecule.

Nucleic Acid Amplification

One aspect of the invention relates to performing a nucleic acidamplification on two or more nucleotide sequences to produce two or moreamplicons in a fluid that is in contact with an array of probes. Theamplification of a nucleotide sequence can be performed by any method ofamplification. Methods of amplification include, for example: polymerasechain reaction (PCR), strand displacement amplification (SDA), andnucleic acid sequence based amplification (NASBA), and Rolling CircleAmplification (RCA).

The polymerase chain reaction (PCR) is widely used and described, andinvolves the use of primer extension combined with thermal cycling toamplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202.In addition, there are a number of variations of PCR which also find usein the invention, including quantitative PCR or Q-PCR, “quantitativecompetitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or “AP-PCR”,“immuno-PCR”, “Alu-PCR”, “PCR single strand conformational polymorphism”or “PCR-SSCP”, allelic PCR, “reverse transcriptase PCR” or “RT-PCR”,“biotin capture PCR”, “vectorette PCR”. “panhandle PCR”, and “PCR selectcDNA subtraction”, among others. Strand displacement amplification (SDA)is generally described in Walker et al., in U.S. Pat. Nos. 5,455,166 and5,130,238. Nucleic acid sequence based amplification (NASBA) isgenerally described in U.S. Pat. No. 5,409,818.

The amplification method can use temperature cycling or be isothermal.The amplification method can be exponential or linear. Foramplifications with temperature cycling, a temperature cycle generallycorresponds to an amplification cycle. Isothermal amplifications can insome cases have amplification cycles, such as denaturing cycles, and inother cases, the isothermal amplification reaction will occurmonotonically with out any specific amplification cycle.

One aspect of the invention performing PCR amplification on two or morenucleotide sequences to produce two or more amplicons in a fluid that isin contact with an array of probes. PCR is used to amplify specificregions, or nucleotide sequences of a DNA strand. This region can be,for example, a single gene, just a part of a gene, or a non-codingsequence. PCR methods typically amplify DNA fragments of up to 10 kilobase pairs (kb), although some techniques allow for amplification offragments up to 40 kb in size.

The PCR generally requires several components: (1) The DNA template thatcontains the region of the nucleic acid sequence to be amplified; (2)one or more primers, which are complementary to the DNA regions at the5′ and 3′ ends of the DNA region that is to be amplified; (3) a DNApolymerase (e.g. Taq polymerase or another DNA polymerase with atemperature optimum at around 70° C.), used to synthesize a DNA copy ofthe region to be amplified; (4) Deoxynucleotide triphosphates, (dNTPs);(5) a buffer solution, which provides a suitable chemical environmentfor optimum activity and stability of the DNA polymerase; and (6) adivalent cation such as magnesium or manganese ions.

Prior to the first cycle, the reaction can be subjected to a hold stepduring an initialization step, the PCR reaction can be heated to atemperature of 94-98° C., and this temperature is then held for 1-9minutes. This first hold is employed to ensure that most of the DNAtemplate and primers are denatured, i.e., that the DNA is melted bydisrupting the hydrogen bonds between complementary bases of the DNAstrands. In some embodiments a hot-start PCR can be utilized.

Temperature cycling can then begin with one step at, for example, 94-98°C. for e.g. 20-30 seconds (denaturation step). The denaturation isfollowed by the annealing step. In this step the reaction temperature islowered so that the primers can anneal to the single-stranded DNAtemplate. The temperature at this step depends on the meltingtemperature of the primers, and is usually between 50-64° C. for e.g.20-40 seconds.

The annealing step is followed by an extension/elongation step duringwhich the DNA polymerase synthesizes new DNA strands complementary tothe DNA template strands. The temperature at this step depends on theDNA polymerase used. Taq polymerase has a temperature optimum of about70-74° C.; thus, a temperature of 72° C. may be used. The DNA polymerasecondenses the 5′-phosphate group of the dNTPs with the 3′-hydroxyl groupat the end of the nascent (extending) DNA strand, i.e., the polymeraseadds dNTP's that are complementary to the template in 5′ to 3′direction, thus reading the template in 3′ to 5′ direction. Theextension time may depend on both on the DNA polymerase used and on thelength of the DNA fragment to be amplified. Thus, in the processdescribed, each temperature cycle has three phases; denaturation,annealing, and elongation. The amplified products from the amplificationare referred to as amplicons. Generally, 10-40 cycles, usually 20-30cycles of PCR are performed.

In some embodiments, the amplification methods of the invention utilizeprimers. A primer is a nucleic acid strand, or a related molecule thatserves as a starting point for DNA replication. A primer is oftenrequired because most DNA polymerases cannot begin synthesizing a newDNA strand from scratch, but can only add to an existing strand ofnucleotides. The primers of the invention are usually short, chemicallysynthesized DNA molecules with a length about 10 to about 30 bases. Thelength of primers can be for example about 20-30 nucleotides, and thesequence of the primers are complementary to the beginning and the endof the DNA fragment to be amplified. They anneal (adhere) to the DNAtemplate at these starting and ending points, where DNA polymerase bindsand begins the synthesis of the new DNA strand.

The design of primers is well known in the art. Pairs of primers shouldgenerally have the similar melting temperatures (Tm). A primer with a Tmsignificantly higher than the reaction's annealing temperature maymishybridize and extend at an incorrect location along the DNA sequence,while Tm significantly lower than the annealing temperature may fail toanneal and extend at all.

In some embodiments of the invention, degenerate primers are used. Theseare actually mixtures of similar, but not identical, primers. Degenerateprimers can be used, for example, when primer design is based on proteinsequence. As several different codons can code for one amino acid, it isoften difficult to deduce which codon is used in a particular case.Therefore primer sequence corresponding to the amino acid isoleucinemight be “ATH”, where A stands for adenine, T for thymine, and H foradenine, thymine, or cytosine, according to the genetic code for eachcodon. Use of degenerate primers can greatly reduce the specificity ofthe PCR amplification.

In some embodiments of the invention, the primers are labeled, and thelabels become incorporated into the amplicons formed by such primers.The primers can be labeled with one or more labels that are for examplefluorescent, quenchers, or members of a FRET pair. Methods of attachingthese moieties to primers is well known in the art.

One aspect of the invention is an array of probes that is in fluidcontact with an amplification reaction in which multiple amplicons areformed with multiple primer sets. The array of probes in fluid contactwith the amplification reaction allows for the quantitation of theamount of amplicon generated at each cycle. As with Q-PCR, the amount ofamplicon generated at each cycle can be plotted against the number ofcycles, which can be used to accurately determine the amount of nucleicacid sequence from which the amplicons were generated.

The measurement of the amount of amplicon during or after each cycle isfacilitated by measuring the hybridization kinetics of the amplicons tothe probes on the array. In some embodiments, the rate of hybridizationof the amplicon to the probe is measured during the annealing phase, thedenaturing phase or the elongation phase. In a three phase PCR,typically, the rate of amplicon hybridization to probe is measuredduring the annealing phase of the PCR temperature cycle, where the rateof going from unbound to bound amplicon can be measured and the rate canbe correlated with the concentration of the analytes in the fluid.Measuring binding during the denaturing phase can allow thedetermination of the “off” rate the amplicons. In some embodiments, morethan 3 temperature phases are used within a temperature cycle, and theseparate temperature phases can be used do define specific hybridizationconditions. For example, as shown in FIG. 11, a 5 phase temperaturecycle can be used. The 5 phase temperature cycle shown in FIG. 11 has 3phases that are similar to the 3 phases of a conventional PCR, and inaddition, there is another denaturing phase followed by a hybridizationphase. In this embodiment, the kinetics of amplicon hybridization ismeasured during the hybridization phase. The 5 phase method allows thehybridization phase to be optimized for measuring amplicon binding,while the annealing phase is optimized for primer annealing. In someembodiments, the temperature of the hybridization phase is lower thanthe temperature of the annealing phase. In some embodiments, thetemperature of the hybridization phase is higher than that of theannealing phase.

In some embodiments, 3, 4, 5, 6 or more phases can be used. For example,in some embodiments, the hybridization phase can comprise multipletemperatures, allowing several hybridization conditions to be measuredduring one amplification cycle. In some cases, the number of phases canvary over the amplification, for example using a 3 phase cycle in somecycles, and a 5 phase cycle in others.

In some embodiments, amplicon hybridization is measured after everycycle. In some embodiments, amplicon hybridization is measured at somebut not all of the amplification cycles. For example, in someembodiments, the first several cycles to about 10 cycles may providelittle information because the level of amplicon is below the detectionthreshold, and in such cases, hybridization at few or none of theearlier cycles may be measured, while in the later cycles, hybridizationat every cycle or almost every cycle may be measured. In contrast, thedata during the exponential phase of amplification can yield the mostuseful data, and in this region, the amplicon hybridization may bemeasured during or after every or almost every cycle. In someembodiments, amplicon hybridization is measured on average during orafter every 2, 3, 4, 5, 6, 7, 8, 9, or 10 amplification cycles.

One aspect of the invention is a method for performing multiplexquantitative PCR comprising (a) measuring the amount of 10 or moreamplicons corresponding to 10 or more different nucleotide sequences ina single fluid volume during or after multiple amplification cycles todetermine amplicon amount-amplification cycle values, and (b) using theamplicon amount-amplification cycle values to determine the presence oramount of the 10 or more nucleotide sequences in a sample. In someembodiments, the invention provides a method of performing multiplex PCRof 20 or more amplicons corresponding to 20 or more different nucleotidesequences are used to determine the amount of 20 or more nucleotidesequences. In some embodiments, the invention provides a method ofperforming multiplex PCR of 50 or more amplicons corresponding to 50 ormore different nucleotide sequences are used to determine the amount of50 or more nucleotide sequences. Multiplex Q-PCR refers to aquantitative PCR reaction wherein the concentration of more than oneamplicon corresponding to more than one nucleic acid sequence ismeasured in the same amplification reaction in the same fluid volume. Itis known in the art, for instance, that real-time PCR or Q-PCR systemscan incorporate detector dyes with different emission wavelengths, suchthat each dye can be detected in the same solution. These prior artmethod can be used for a small number of different amplicons, but arelimited by the overlap of the fluorescent emission spectra. It is alsoknown in the art, that where there are a larger number of amplicons, tosplit the sample into a number of different wells, for instance on aplate, and perform Q-PCR on the plate at one time in order to amplifythe sample in each well of the plate. The present invention allows forlarger numbers of amplicons, and therefore a larger number of nucleicacid sequences to be measured in the same amplification reaction in thesame fluid. The present invention allows for example for a multiplexquantitative PCR reaction of greater that 10, 20, 50, 100, 200, 500,1,000, 2,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000 or morenucleic acid sequences in the same amplification reaction in the samefluid.

One aspect of the methods of the present invention includes the step ofperforming an algorithm on real-time binding data to determinecross-hybridization of amplicons for multiple probes on a substrate. Oneembodiment involves improving the quality of analyte-probe bindingmeasurements by determining and correcting for cross-hybridization.Algorithms for determining cross-hybridization are described in U.S.patent Ser. No. 11/758,621, incorporated herein by reference.

Arrays

One aspect of the invention is an array that has a solid surface with aplurality of probes attached to it, where the array can be used for thereal-time measurement of binding of amplicons to the plurality ofprobes.

The arrays of the present invention comprise probes attached to a solidsubstrate. The solid substrate may be biological, nonbiological,organic, inorganic, or a combination of any of these, existing asparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides,semiconductor integrated chips, etc. The solid substrate is preferablyflat but may take on alternative surface configurations. For example,the solid substrate may contain raised or depressed regions on whichsynthesis or deposition takes place. In some embodiments, the solidsubstrate will be chosen to provide appropriate light-absorbingcharacteristics. For example, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, Gap, SiO₂,SiN₄, modified silicon, or any one of a variety of gels or polymers suchas (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, or combinations thereof.

The substrate can be a homogeneous solid and/or unmoving mass muchlarger than the capturing probe where the capturing probes are confinedand/or immobilized within a certain distance of it. The mass of thesubstrate is generally at least 100 times larger than capturing probesmass. In certain embodiments, the surface of the substrate is planarwith roughness of 0.1 nm to 100 nm, but typically between 1 nm to 100nm. In other embodiments the substrate can be a porous surface withroughness of larger than 100 nm. In other embodiments, the surface ofthe substrate can be non-planar. Examples of non-planar substrates arespherical magnetic beads, spherical glass beads, and solid metal and/orsemiconductor and/or dielectric particles. As used herein, the termarray and the term microarray are used interchangeably.

In some embodiments the substrate is optically clear, allowing light tobe transmitted through the substrate, and allowing excitation and ordetection to occur from light passing through the substrate. In someembodiments the substrate is opaque. In some embodiments, the substrateis reflective, allowing for light to pass through the surface layercontaining probes and reflect back to a detector.

In some embodiments, the array is incorporated into the fluid containerin which the amplification reaction will take place. In some cases, thearray is part of the wall or base of the container. In some cases thearray mates with other elements, forming a seal, and creating a fluidcontainer for carrying out the amplification reaction.

In some embodiments, glass slides are used to prepare biochips. Thesubstrates (such as films or membranes) can also be made of silica,silicon, plastic, metal, metal-alloy, anopore, polymeric, and nylon. Thesurfaces of substrates can be treated with a layer of chemicals prior toattaching probes to enhance the binding or to inhibit non-specificbinding during use. For example, glass slides can be coated withself-assembled monolayer (SAM) coatings, such as coatings of asaminoalkyl silanes, or of polymeric materials, such as acrylamide andproteins. A variety of commercially available slides can be used. Someexamples of such slides include, but are not limited to, 3D-link®(Surmodics), EZ-Rays® (Mosaic Technologies), Fastslides® (Schleicher andSchuell), Superaldehyde®, and Superamine® (CEL Technologies).

Probes can be attached covalently to the solid surface of the substrate(but non-covalent attachment methods can also be used).

A number of different chemical surface modifiers can be added tosubstrates to attach the probes to the substrates. Examples of chemicalsurface modifiers include N-hydroxy succinimide (NHS) groups, amines,aldehydes, epoxides, carboxyl groups, hydroxyl groups, hydrazides,hydrophobic groups, membranes, maleimides, biotin, streptavidin, thiolgroups, nickel chelates, photoreactive groups, boron groups, thioesters,cysteines, disulfide groups, alkyl and acyl halide groups, glutathiones,maltoses, azides, phosphates, and phosphines. Glass slides with suchchemically modified surfaces are commercially available for a number ofmodifications. These can easily be prepared for the rest, using standardmethods (Microarray Biochip Technologies, Mark Schena, Editor, March2000, Biotechniques Books).

In one embodiment, substrate surfaces reactive towards amines are used.An advantage of this reaction is that it is fast, with no toxicby-products. Examples of such surfaces include NHS-esters, aldehyde,epoxide, acyl halide, and thio-ester. Most proteins, peptides,glycopeptides, etc. have free amine groups, which will react with suchsurfaces to link them covalently to these surfaces. Nucleic acid probeswith internal or terminal amine groups can also be synthesized, and arecommercially available (e.g., from IDT or Operon). Thus, nucleic acidscan be bound (e.g., covalently or non-covalently) to surfaces usingsimilar chemistries.

The substrate surfaces need not be reactive towards amines, but manysubstrate surfaces can be easily converted into amine-reactivesubstrates with coatings. Examples of coatings include amine coatings(which can be reacted with bis-NHS cross-linkers and other reagents),thiol coatings (which can be reacted with maleimide-NHS cross-linkers,etc.), gold coatings (which can be reacted with NHS-thiol cross linkers,etc.), streptavidin coatings (which can be reacted with bis-NHScross-linkers, maleimide-NHS cross-linkers, biotin-NHS cross-linkers,etc.), and BSA coatings (which can be reacted with bis-NHScross-linkers, maleimide-NHS cross-linkers, etc.). Alternatively, theprobes, rather than the substrate, can be reacted with specific chemicalmodifiers to make them reactive to the respective surfaces.

A number of other multi-functional cross-linking agents can be used toconvert the chemical reactivity of one kind of surface to another. Thesegroups can be bifunctional, tri-functional, tetra-functional, and so on.They can also be homo-functional or hetero-functional. An example of abi-functional cross-linker is X—Y—Z, where X and Z are two reactivegroups, and Y is a connecting linker. Further, if X and Z are the samegroup, such as NHS-esters, the resulting cross-linker, NHS—Y—NHS, is ahomo-bi-functional cross-linker and would connect an amine surface withan amine-group containing molecule. If X is NHS-ester and Z is amaleimide group, the resulting cross-linker, NHS—Y-maleimide, is ahetero-bi-functional cross-linker and would link an amine surface (or athiol surface) with a thio-group (or amino-group) containing probe.Cross-linkers with a number of different functional groups are widelyavailable. Examples of such functional groups include NHS-esters,thio-esters, alkyl halides, acyl halides (e.g., iodoacetamide), thiols,amines, cysteines, histidines, di-sulfides, maleimide, cis-diols,boronic acid, hydroxamic acid, azides, hydrazines, phosphines,photoreactive groups (e.g., anthraquinone, benzophenone), acrylamide(e.g., acrydite), affinity groups (e.g., biotin, streptavidin, maltose,maltose binding protein, glutathione, glutathione-S-transferase),aldehydes, ketones, carboxylic acids, phosphates, hydrophobic groups(e.g., phenyl, cholesterol), etc. Such cross-linkers can be reacted withthe surface or with the probes or with both, in order to conjugate aprobe to a surface.

Other alternatives include thiol reactive surfaces such as acrylate,maleimide, acyl halide and thio-ester surfaces. Such surfaces cancovalently link proteins, peptides, glycopeptides, etc., via a (usuallypresent) thiol group. Nucleic acid probes containing pendantthiol-groups can also be easily synthesized.

Alternatively, one can modify glass surfaces with molecules such aspolyethylene glycol (PEG), e.g. PEGs of mixed lengths.

Other surface modification alternatives (such as photo-crosslinkablesurfaces and thermally cross-linkable surfaces) are known to thoseskilled in the art. Some technologies are commercially available, suchas those from Mosiac Technologies (Waltham, Mass.), Exiqon™ (Vedbaek,Denmark), Schleicher and Schuell (Keene, N.H.), Surmodics™ (St. Paul,Minn.), Xenopore™ (Hawthorne, N.J.), Pamgene (Netherlands), Eppendorf(Germany), Prolinx (Bothell, Wash.), Spectral Genomics (Houston, Tex.),and Combimatrix™ (Bothell, Wash.).

Surfaces other than glass are also suitable for such devices. Forexample, metallic surfaces, such as gold, silicon, copper, titanium, andaluminum, metal oxides, such as silicon oxide, titanium oxide, and ironoxide, and plastics, such as polystyrene, and polyethylene, zeolites,and other materials can also be used. The devices can also be preparedon LED (Light Emitting Diode) and OLED (Organic Light Emitting Diode)surfaces. An array of LEDs or OLEDs can be used at the base of a probearray. An advantage of such systems is that they provide easyoptoelectronic means of result readout. In some cases, the results canbe read-out using a naked eye.

Probes can be deposited onto the substrates, e.g., onto a modifiedsurface, using either contact-mode printing methods using solid pins,quill-pins, ink-jet systems, ring-and-pin systems, etc. (see, e.g., U.S.Pat. Nos. 6,083,763 and 6,110,426) or non-contact printing methods(using piezoelectric, bubble-jet, syringe, electro-kinetic, mechanical,or acoustic methods. Devices to deposit and distribute probes ontosubstrate surfaces are produced by, e.g., Packard Instruments. There aremany other methods known in the art. Preferred devices for depositing,e.g., spotting, probes onto substrates include solid pins or quill pins(Telechem/Biorobotics).

The arrays of the present invention can also be three-dimensional arrayssuch as porous arrays. Such as devices consisting of one or more porousgel-bound probes in an array or an array of arrays format. A device canhave one or more such structures and the structures can be of anygeometric shape and form. The structures can also be verticallystraight, angled, or twisted. Thus, each device denotes a (multiplexed)reaction site. The device can be used to perform reactionssimultaneously or sequentially. Any of the known substrates andchemistries can be used to create such a device. For example, glass,silica, silicon wafers, plastic, metals; and metal alloys can all beused as the solid support (see. e.g., Stillman B A, Tonkinson J L,Scleicher and Schuell; Biotechniques, 29(3), 630-635, 2000; Rehmna et.al; Mosaic Technologies Inc., Nucleic Acids Research, 27(2), 649-655,1999). In other embodiments, the intermediate species can be immobilizedto the substrate using mechanical and/or electrostatic and/or andmagnetic forces. Examples are magnetic beads with magnetic fields andglass beads with electrostatic fields. Bead based methods are described,for example in Gunderson et al., Genome Research, 870-877, 2004.

In other embodiments, the microarrays are manufactured through thein-situ synthesis of the probes. This in-situ synthesis can be achievedusing phosphoramidite chemistry and/or combinatorial chemistry. In somecases, the deprotection steps are performed by photodeprotection (suchas the Maskless Array Synthesizer (MAS) technology, (NimbleGen, or thephotolithographic process, by Affymetrix). In other cases, deprotectioncan be achieved electrochemically (such as in the Combimatrixprocedure). Microarrays for the present invention can also bemanufactured by using the inkjet technology (Agilent).

For the arrays of the present invention, the plurality of probes arelocated on multiple addressable regions on the solid substrate. In someembodiments the solid substrate has about 2, 3, 4, 5, 6, or 7-10, 10-50,50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000,50,000-100,000, 100,000-500,000, 500,000-1,000,000 or over 1,000,000addressable regions with probes.

The spots may range in size from about 1 nm to 10 mm, in someembodiments from about 1 to 1000 micron and more in some embodimentsfrom about 5 to 100 micron. The density of the spots may also vary,where the density is generally at in some embodiments about 1spot/cm.sup.2, in some embodiments at least about 100 spots/cm.sup.2 andin other embodiments at least about 400 spots/cm.sup.2, where thedensity may be as high as 10.sup.6 spots/cm.sup.2 or higher.

The shape of the spots can be square, round, oval or any other arbitraryshape.

In some embodiments, the optical signal moiety, for example, afluorescent moiety is bound directly to the surface, but is notcovalently bound to a probe, and in these cases the probe need not belabeled. The fluorescent moiety can be bound to the surface orsynthesized in-situ by any of the methods described above for probes.The fluorescent moiety can be attached to an oligonucleotide that is nota probe, for example, having a sequence that is not complementary totarget amplicons in solution.

In some embodiment, a fluorescent moiety on the surface (surface-boundlabel) can be brought to the proximity of the probe viapost-probe-synthesis or post-probe-deposition methods.

In some embodiments, the label can be bound to the probe by non-covalentmeans, such as by hybridization. For example, in certain embodiments ofthe present invention, some or all of the probes on the microarray maycontain two different sequence segments: one segment that consists of asequence that is specific to the probe and specific for the detection ofa given target amplicon, and another segment that is a sequence that iscommon to all or many of the probes on the microarray. These twosequence segments can be immediately adjacent to each other on theprobe, or separated by a linker. In this embodiment, the microarray isfirst hybridized with a (labeled oligonucleotide that is complementaryto the common sequence segment, thus resulting in a microarray in whichthe spots or features where the probes are located also now containfluorescent labels. These non-covalently bound labels can be bound tothe probe such that FRET and or quenching of the label occurs uponbinding of an amplicon to the specific portion of the probe. This methodcan be advantageous, for instance by (1) lowering the cost ofmanufacturing microarrays that can be used in the real-time platformand/or (2) enabling the use of in-situ synthesized arrays in the realtime platform. The labeled oligonucleotide can be a locked nucleic acid(LNA) oligonucleotide. LNA oligonucleotides can be useful because theLNA modification can result in enhanced hybridization properties (forexample, diminishing the sequence length that is needed to achieve acertain Tm) (Jepsen et al., Oligonucleotides. 2004; 14(2):130-46).

Systems

One aspect of the invention is a system comprising: (a) a PCRamplification reaction chamber capable of receiving: (i) a substratecomprising a surface with an array of nucleic acid probes atindependently, addressable locations, and (ii) a fluid to be heldcontact with the substrate, the fluid comprising a nucleic acid samplecomprising multiple nucleotide sequences, primers, and enzymes; (b) atemperature controller capable of carrying out multiple PCR temperaturephases and temperature cycles comprising: (i) a heating and coolingmodule for raising and lowering the temperature of the fluid and/or thesubstrate; and (ii) a temperature sensor; and (c) a detector capable ofdetecting light signals as a function of time from the independentlyaddressable locations on the substrate within the chamber at a specificphase or phases during or after a plurality of temperature cycles whilethe fluid is in contact with the substrate. In some embodiments, thesystem further comprises: (d) an analysis block comprising a computerand software capable of determining the amounts of amplified productshybridized to the array of probes using the detected light as a functionof time, and of determining the amounts of multiple nucleotide sequencesin a sample using the amounts of amplified products determined during orafter a plurality of temperature cycles.

The fluid volume can be introduced and held in the system by any methodthat will maintain the fluid in contact with the solid support. In manycases the fluid is held in a chamber. In some embodiments the chamber isopen on one face, in other embodiments the chamber will mostly enclosethe fluid. In some embodiments, the chamber will have one or more portsfor introducing and/or removing material (usually fluids) from thechamber. In some embodiments one side of the chamber comprises the solidsubstrate on which the probes are attached. In some embodiments thechamber is integral to the solid substrate. In some embodiments, thechamber is a sub-assembly to which the solid substrate with probes canbe removably attached. In some embodiments, some or all of the fluidchamber is an integral part of the instrument that comprises thedetector. The chamber can be designed such that the signal that can becorrelated with amplicon-probe binding can be detected by a detectoroutside of the chamber. For instance, all or a portion of the chambercan be transparent to light to allow light in or out of the chamber tofacilitate excitation and detection of fluorophores.

The system can incorporate one or more microfluidic devices.Microfluidic devices are fluid systems in which the volumes of fluid aresmall, typically on the order of microliters to nanoliters. In someembodiments the microfluidics can handle tens to thousands of samples insmall volumes. Microfluidics used in the invention can be active orpassive. By using active elements such as valves in the microfluidicdevice, microfluidic circuits can be created. This allows not only theuse of small reagent volumes but also a high task parallelization sinceseveral procedures can be processed and physically be fitted on the samechip.

In microfluidic channels the flow of liquid can be completely laminar,that is, all of the fluid moves in the same direction and at the samespeed. Unlike turbulent flow this allows the transport of molecules inthe fluid to be very predictable. The microfluidic devices useful in theinvention can be made of glass or plastic. In some embodiments,polydimethylsiloxane (PDMS), a type of silicone can be used. Someadvantages of PDMS are that it is inexpensive, optically clear andpermeable to several substances, including gases. In some embodimentssoft lithography or micromolding can be used to create PDMS basedmicrofluidic devices. The devices can use pressure driven flow,electrodynamic flow, or wetting driven flow.

In some embodiments the microfluidic device has multiple chambers, eachchamber having a real-time microarray. In some embodiments, the array isincorporated into the microfluidic device. In some embodiments, themicrofluic device is formed by adding features to a planar surfacehaving multiple real-time microarrays in order to create chamberswherein the chamber correspond to the real-time microarray. In someembodiments, a substrate having 3-dimensional features, for example aPDMS surface with wells and channels, is placed in contact with asurface having multiple real-time microarrays to form a microfluidicdevice having multiple arrays in multiple chambers.

A device having multiple chambers, each with a real-time microarray canbe used in order to analyze multiple samples simultaneously. In someembodiments, multiple chambers have sample fluid derived from the samesample. Having sample fluid from the same sample in multiple chamberscan be useful, for example to measure each with different arrays foranalyzing different aspects of the same sample, or for example forincreasing accuracy by parallel measurements on identical arrays. Insome cases, different amplicons within the same sample will havedifferent optimum temperature profile conditions. Thus, in someembodiments, the same sample is divided into different fluid volumes,and the different fluid volumes are in different chambers with real-timemicroarrays; and at least some of the different fluid volumes are givena different temperature cycle.

In some embodiments, multiple chambers have sample fluid from differentsources. Having sample fluid from different sources can be useful inorder to increase throughput by measuring more samples in a given timeperiod on a given instrument. In some embodiments, the microfluidic withmultiple chambers containing real-time microarrays can be used fordiagnostic applications. The device may have about 2, 3, 4, 5, 6, 7, 8,9, 10, 10-15, 15-20, 20-30, 30-50, 50-75, 75-100, or more than 100chambers each having a real time microarray.

The detector assembly can comprise a single detector or an array ofdetectors or transducers. As used herein, the terms detector andtransducer are used interchangeably, and refer to a component that iscapable of detecting a signal that can be correlated with the amount ofamplicon-probe binding. Where the detector system is an array oftransducers, in some embodiments, the detector system is a fixed arrayof transducers, wherein one or more transducers in the transducer arraycorresponds to one independently addressable area of the array. In someembodiments, the detector or the array of transducers scans the arraysuch that a given detector or transducer element detects signals fromdifferent addressable areas of the array during a binding reaction.

In some embodiments the detector array is in contact with the solidsubstrate. In some embodiments, the detector is at a distance away fromthe substrate. Where the detector is a distance away from the substrate,in some embodiments, the detector or detector array is capable ofscanning the substrate in order to measure signal from multipleaddressable areas. In some embodiments, the detector is an opticaldetector which is optically coupled to the substrate. The detector canbe optically coupled to the substrate, for example with one or morelenses or waveguides.

FIG. 12 shows an example of a real-time microarray system where thedetection system comprises a sensor array in intimate proximity of thecapturing spots. In this embodiment, individual sensors detect thebinding events of a single capturing spots.

In some embodiments, the detector is optically coupled through spatiallyconfined excitation. This method is useful to optically couple thesubstrate to detector for a small region of substrate with probes. Thismethod generally requires only a single detector, since only one regioncan create signal at a time. The method can be used in scanning systems,and is applicable in assays which an excitation is required fordetection, such as fluorescence spectroscopy or surface plasmonresonance (SPR) methods.

In some embodiments, the detector is optically coupled through imagingusing focal plane detector arrays: In this method the signal generatedfrom the system is focused on a focal point detector array. Thisapproach useful for optical detection systems where signal focusing canbe carried out using lenses and other optical apparatus. Examples ofdetectors in these embodiments are complementary metal oxidesemiconductor (CMOS) and charge coupled device (CCD) image sensors.

In some embodiments, the detector is optically coupled through surfaceimaging: In this method the detectors are placed in intimate proximityof the capturing probes such that the signal generated from thecapturing region can only be observed by the dedicated detector. If amicroarray with multiple capturing spots is used, multiple detectors areused, each dedicated to an individual spot. This method can be used inelectrochemical-, optical-, and magnetic-based biosensors.

In some embodiments, the detector is optically coupled through surfaceimaging using signal couplers: In this method the detectors are notplace in proximity of the capturing spots, however a signal coupler isused to direct signal from the capturing region to a detector. Thismethod is generally used in optical detection systems where the signalcoupling elements is a plurality of optical waveguide. Examples ofsignal coupling elements include fiber optic cables, fiber opticbundles, fiber optic faceplates, and light pipes.

Were a microfluidic device with multiple chambers, each chambercontaining a real-time microarray is used, the instrument can have onedetection device that measures multiple arrays, or each chamber can haveits own detector or its own set of detectors.

The detectors of the present invention must be capable of capturingsignal at multiple time points in real time, during the bindingreaction. In some embodiments the detector is capable of measuring atleast two signals in less than about 1 psec, 5 psec, 0.01 nsec, 0.05nsec, 0.1 nsec, 0.5 nsec, 1 nsec, 5 nsec, 0.01 μsec, 0.05 μsec, 0.1μsec, 0.5 μsec, 1 μsec, 5 μsec, 0.01 msec, 0.05 msec, 0.1 msec, 0.5msec, 1 msec, 5 msec, 10 msec, 50 msec, 100 msec, 0.5 sec, 1 sec, 5 sec,10 sec, or 60 sec.

In some embodiments the detector detects the signal at the substrate. Insome embodiments the detector will detect the signal in the solution. Insome embodiments, the detector will detect signal in both the solutionand at the substrate.

In some embodiments the detector system is capable of detectingelectrical, electrochemical, magnetic, mechanical, acoustic, orelectromagnetic (light) signals.

Where the detector is capable of detecting optical signals, the detectorcan be, for example a photomultiplier tube (PMT), a CMOS sensor, or a(CCD) sensor. In some embodiments, the detector comprises a fiber-opticsensor.

In some embodiments, the system comprising the detector is capable ofsensitive fluorescent measurements including synchronous fluorimetry,polarized fluorescent measurements, laser induced fluorescence,fluorescence decay, and time resolved fluorescence.

In some embodiments, the system comprises a light source, for example,for excitation of fluorescence. The light source is generally opticallycoupled to the substrate, for example with one or more lenses orwaveguides. The light source can provide a single wavelength, e.g. alaser, or a band of wavelengths.

FIG. 13 shows a block diagram of the components of an embodiment of amultiplex Q-PCR system of the present invention. The system comprises of(a) reaction chamber which includes the microarray substrate with probeswhere the probes are designed for binding with a multiplicity ofamplicons generated in the amplification reaction in the chamber (here,probes A, B, and C, on separate addressable locations are capable ofspecifically binding to amplicons A, B, and C respectively); (b) atemperature controller comprising: (i) a heating and cooling module and(ii) a temperature sensor; and (c) detector, the detector optionallyconnected to (d) an analysis block, where the latter is a part of acomputing system.

FIG. 14 shows an example of a real-time microarray system wherereal-time binding of BHQ2 quencher-labeled cDNA molecules are detectedusing a fluorescent laser-scanning microscope. The substrate in thisexample is a transparent glass slides and the probes are 25 bpCy5-labeled oligonucleotides. In this embodiment, the light source(laser) and detector are both located on the back of the substrate.

In some embodiments, the system comprises an instrument that can accepta sub-assembly. The sub assembly comprises a chamber that will hold thefluid volume and the solid substrate having a surface and a plurality ofprobes. The sub-assembly can be loaded into the instrument in order tomonitor the hybridization of amplicons to probes in real time.

In some embodiments, the system comprises: an assay assembly comprisingmeans to engage a microarray and means to perform an assay on a surfaceof the microarray; and a detector assembly comprising means to detectsignals measured at multiple time points from each of a plurality ofspots on the microarray during the performance of the assay.

In some embodiments, the means to perform the assay comprise acompartment wherein the surface of the microarray comprises a floor ofthe compartment and means to deliver reagents and analytes into thecompartment. Any method can be used to seal the microarray to thecompartment including using adhesives and gaskets to seal the fluid. Anymethod can be used to deliver reagents and analytes including usingsyringes, pipettes, tubing, and capillaries.

In some embodiments, the system comprises a means of controlling thetemperature. Control of temperature can be important to allow control ofbinding reaction rates, e.g. by controlling stringency. The temperaturecan be controlled by controlling the temperature at any place within thesystem including controlling the temperature of the fluid or thetemperature of the solid substrate. Any means can be used forcontrolling the temperature including resistive heaters, Peltierdevices, infrared heaters, fluid or gas flow. The temperatures can bethe same or different for solution or substrate or different parts ofeach. Ideally the temperature is consistently controlled within thebinding region. In some embodiments the temperature is controlled towithin about 0.01, 0.05, 0.1, 0.5, or 1° C. In some embodiments, forexample for a PCR amplification reaction, the temperature is controlledto within 0.1° C.

In some embodiments the system is capable of changing the temperatureduring the amplification in order to define the phases of thetemperature cycles. In some embodiments, the temperature can be rapidlychanged during the amplification reaction. In some embodiments, thesystem is capable of changing the temperature at a rate of temperaturechange corresponding to a change of 1° C. in less than about 0.01 msec,0.1 msec, 0.5 msec, 1 msec, 5 msec, 10 msec, 50 msec, 100 msec, 0.5 sec,1 sec, 10 sec, or 60 sec. In some embodiments, for example for a PCRamplification reaction, the system is capable of changing temperature ata rate of greater than about 5° C., 10° C., or 20° C. per second.

In other embodiments the temperature is changed slowly, graduallyramping the temperature over the course of the binding reaction.

One exemplary embodiment of changing the temperature involves a changein temperature to change the binding stringency and probability. Bychanging temperature we can alter the stringency and observe thecapturing the new capturing process with a new set of capturingprobabilities.

In some embodiments, the system is capable of measuring temperature inone or multiple locations in the solution or on the solid substrate. Thetemperature can be measured by any means including, for example, bythermometer, thermocouple, or thermochromic shift.

In some embodiments the system comprises a feedback loop for temperaturecontrol wherein the measured temperature is used as an input to thesystem in order to more accurately control temperature.

In some embodiments, the system comprises an apparatus to add or removematerial from the fluid volume. In some embodiments, the system can addor remove a liquid from the fluid volume. In some embodiments, thesystem is capable of adding or removing material from the fluid volumein order to change the: concentration, pH, stringency, ionic strength,or to add or remove a competitive binding agent. In some embodiments,the system is capable of changing the volume of the fluid volume.

One exemplary embodiment of adding material to the fluid volumecomprises the addition of incubation buffer. By adding the incubationbuffer, the concentration of analytes in the system will decrease andtherefore the binding probability and kinetic of binding will bothdecrease. Furthermore, if the reaction has already reached equilibrium,the addition of the buffer will cause the system to move anotherequilibrium state in time.

Another exemplary embodiment of adding material to the fluid volume isadding a competing binding species. The competing species can be of thesame nature of the analyte but in general they are molecules which haveaffinity to capturing probes. For DNA microarrays for example, thecompeting species can be synthesized DNA oligo-nucleotides withpartially or completely complementary sequence to the capturing probes.In immunoassays, the competing species are antigens.

In some embodiments the system comprises elements to apply an electricpotential to the fluid volume to electrically change the stringency ofthe medium. In some embodiments, the system will provide an electricalstimulus to the capturing region using an electrode structure which isplaced in proximity of the capturing region. If the analyte is anelectro-active species and/or ion, the electrical stimulus can apply anelectrostatic force of the analyte. In certain embodiments, thiselectrostatic force is adjusted to apply force on the bonds betweenanalyte and capturing probe. If the force is applied to detach themolecule, the affinity of the analyte-probe interaction is reduced andthus the stringency of the bond is evaluated. The electrical stimulus isgenerally a DC and/or time-varying electrical potentials. Theiramplitude can be between 1 mV to 10 V, but typically between 10 mV to100 mV. The frequency of time-varying signal can be between 1 Hz to 1000MHz, in some embodiments, the frequency of the time-varying signal isbetween 100 Hz to 100 kHz. The use of electric potential to controlstringency is described in U.S. Pat. No. 6,048,690.

In some embodiments the system comprises a computing system foranalyzing the detected signals. In some embodiments, the system iscapable of transferring time point data sets to the computing systemwherein each time point data set corresponds to detected signal at atime point, and the computing system is capable of analyzing the timepoint data sets, in order to determine a property related to the analyteand probe. The methods of the current invention can, in some cases,generate more data, sometimes significantly more data than forconventional microarrays. Thus a computer system and software that canstore and manipulate the data (for instance, images taken at timepoints) can be essential components of the system. The data can beanalyzed in real-time, as the reaction unfolds, or may be stored forlater access.

The information corresponding to detected signal at each time point canbe single values such as signal amplitude, or can be more complexinformation, for instance, where each set of signal informationcorresponds to an image of a region containing signal intensity valuesat multiple places within an addressable location.

The property related to analyte and/or probe can be, for example,analyte concentration, binding strength, or competitive binding, andcross-hybridization.

In some embodiments the computing system uses algorithms, for examplethe algorithms described herein and in U.S. patent application Ser. No.11/758,621 for determining concentration and/or cross-hybridization.

One aspect of the invention is software for use in performing thecalculation of the amount of nucleotide sequences in the sample from theinformation on the change in optical signal as a function ofamplification cycle. The software is capable of, for example, ofcalculating the CT value, including, for instance, fitting theexponential portion of the curve, determining the background threshold.The software is capable of carrying out these calculations efficientlyfor large numbers of amplicons and nucleotide sequences.

In some embodiments, the system has software for interfacing with theinstrument, for example allowing the user to display information inreal-time and allowing for user to interact with the reaction (i.e., addreagents, change the temperature, change the pH, dilution, etc.).

Uses

The methods and systems of the present invention can be used to measurenucleotide sequences in a variety of sample types including cDNA,genomic DNA, RNA, cells, or viruses.

The methods and systems of the present invention are useful for thedetermination of the presence and amount of multiple nucleotidesequences in a sample. Where the probe and analyte are nucleic acids,the present invention provides methods of expression monitoring and formeasuring genetic information. The invention allows for many nucleotidesequences relating to genes, e.g. 10, 100, 1,000, 10,000, 100,000, ormore genes to be analyzed at once. The term expression monitoring isused to refer to the determination of levels of expression ofparticular, typically preselected, genes. For example, amplicons derivedfrom nucleic acid samples such as messenger RNA that reflect the amountof expression of genes are hybridized to the arrays during or afteramplification cycles, and the resulting hybridization signal as afunction of time provides an indication of the amount of amplicon whichcan then be used to determine the level of expression of each gene ofinterest. In some cases, the whole transcriptome can be measuredcomprising all or a substantial portion of the expression in a cell, orgroup of cells. In some embodiments the expression of only a few genes,such as 5 to 100 genes is measured, for example, to diagnose a specificcondition. In some embodiments, the array has a high degree of proberedundancy (multiple probes per gene) the expression monitoring methodsprovide accurate measurement and do not require comparison to areference nucleic acid.

The methods and systems of this invention may be used in a wide varietyof circumstances including detection of disease, identification ofdifferential gene expression between two samples (e.g., a pathologicalas compared to a healthy sample), screening for compositions thatupregulate or downregulate the expression of particular genes, and soforth. They can be used for the analysis of genetic DNA includingdetermination of single-nucleotide polymorphisms (SNPs) for genotypingand allele discrimination assays and for the detection of DNA and RNAviruses. The methods and systems of the invention can also be used forthe detection of genetically modified organisms (GMO).

The methods and systems of the invention can be used for genetic testingand diagnostics. They can be used for example for newborn screening(e.g. for phenylketonuria or congenital hypothyroidism; diagnostictesting (such as to diagnose or rule out a specific genetic orchromosomal condition or to confirm a diagnosis when a particularcondition is suspected based on physical mutations and symptoms);carrier testing: (e.g. to identify people who carry one copy of a genemutation that, when present in two copies, predictive and presymptomatictesting: (For example, testing for BRCA1 in relation to the risk ofbreast cancer); forensic testing (e.g. to identify an individual forlegal purposes, e.g. to identify crime or catastrophe victims, rule outor implicate a crime suspect, or establish biological relationshipsbetween people (for example, paternity); or for research testing (e.g.finding unknown genes, learning how genes work and advancing ourunderstanding of genetic conditions).

EXAMPLES Example 1

This example demonstrates the use of a real-time microarray to measurethe binding of multiple analytes to multiple probes.

FIG. 15 shows the layout of a 6×6 DNA microarray. Three different DNAprobes (1, 2, and Control) with three different concentrations (2 μM, 10μM, and 20 μM) are spotted and immobilized on the surface asillustrated. The probes contain a single Cy3 fluorescent molecule at the5′ end. The DNA targets in this experiment contain a quencher molecule.The analyte binding in this system results in quenching of fluorescentmolecules in certain spots. FIG. 16 shows a few samples of the real-timemeasurements of the microarray experiment wherein the control targetsare added to the system. As illustrated in FIG. 16, the spots arequenched due to analyte binding.

FIGS. 17-20 each show data for 4 different spots with similaroligonucleotide capturing probes. The target DNA analyte is introducedin the system at time zero and quenching (reduction of signal) occursonly when binding happens. For FIG. 17, the light intensity coefficientof variation was about 15%, however the estimated time constant ratefrom real-time measurements had only 4.4% variations. For FIG. 18 thelight intensity coefficient of variation was about 15%, however theestimated time constant rate from real-time measurements had only 2.1%variations. For FIG. 19 the light intensity coefficient of variation wasabout 22%, however the estimated time constant rate from real-timemeasurements had only 6% variations. For FIG. 20 the light intensitycoefficient of variation was about 22%, however the estimated timeconstant rate from real-time measurements had only a 4.8% variation.

In FIG. 21, the signals measured during two real-time experimentswherein target 2 is applied to the microarray, first at 2 ng and then at0.2 ng, are shown. The measured light intensities at the correspondingprobe spots decay over time as the targets to the probes bind and thequenchers come in close proximity to the fluorescent labels attached tothe end of the probes. The rate of the decay, which can be estimated bya curve fitting technique, is proportional to the amount of the targetpresent. The time constant of the measured process is defined as theinverse of the rate of decay. The ratio of the time constants of the twoprocesses is 10, which is precisely the ratio of the amounts of targetsapplied in the two experiments.

Example 2

This example provides a derivation of an algorithm, and the use of thealgorithm to determine analyte concentration, such as ampliconconcentration, from a real-time binding data. The derivation proceeds asfollows:

Assume that the hybridization process starts at t=0, and considerdiscrete time intervals of the length Δt. Consider the change in thenumber of bound target molecules during the time interval (iΔt,(i+1)Δt). We can write

n _(b)(i+1)−n _(b)(i)=[n _(t) −n _(b)(i)]p _(b)(i)Δt−n _(b)(i)p_(r)(i)Δt,

where n_(t) denotes the total number of target molecules, n_(b)(i) andn_(b)(i+1) are the numbers of bound target molecules at t=iΔt andt=(i+1)Δt, respectively, and where p_(b)(i) and p_(r)(i) denote theprobabilities of a target molecule binding to and releasing from acapturing probe during the i^(th) time interval, respectively. Hence,

$\begin{matrix}{\frac{{n_{b}\left( {i + 1} \right)} - {n_{b}(i)}}{\Delta \; t} = {{\left\lbrack {n_{t} - {\underset{\bullet}{n_{b}}(i)}} \right\rbrack {p_{b}(i)}} - {{n_{b}(i)}{{p_{r}(i)}.}}}} & (1)\end{matrix}$

It is reasonable to assume that the probability of the target releasedoes not change between time intervals, i.e., p_(r)(i)=p_(r), for all i.On the other hand, the probability of forming a target-probe pairdepends on the availability of the probes on the surface of the array.If we denote the number of probes in a spot by n_(p), then we can modelthis probability as

$\begin{matrix}{{{p_{b}(i)} = {{\left( {1 - \frac{n_{b}(i)}{n_{p}}} \right) - p_{b}} = {\frac{n_{p} - {n_{b}(i)}}{n_{p}}p_{b}}}},} & (2)\end{matrix}$

where p_(b) denotes the probability of forming a target-probe pairassuming an unlimited abundance of probes.

By combining (1) and (2) and letting Δt→0, we arrive to

$\begin{matrix}\begin{matrix}{\frac{n_{b}}{t} = {{\left( {n_{t} - n_{b}} \right)\frac{n_{p} - n_{b}}{n_{p}}p_{p}} - {n_{b}p_{r}}}} \\{= {{n_{t}p_{b}} - {\left\lbrack {{\left( {1 + \frac{n_{t}}{n_{p}}} \right)p_{b}} + p_{r}} \right\rbrack n_{b}} + {\frac{p_{b}}{n_{p}}n_{b^{2}}}}}\end{matrix} & (3)\end{matrix}$

Note that in (3), only n_(b)=n_(b)(t), while all other quantities areconstant parameters, albeit unknown. Before proceeding any further, wewill find it useful to denote

$\begin{matrix}{{\alpha = {{\left( {1 + \frac{n_{t}}{n_{p}}} \right)p_{b}} + p_{r}}},{\beta = {n_{t}p_{b}}},{\gamma = {\frac{p_{b}}{n_{p}}.}}} & (4)\end{matrix}$

Clearly, from (4),

${p_{b} = \frac{\beta}{n_{t}}},{n_{p} = \frac{p_{b}}{\gamma}},{p_{r} = {\alpha - {\left( {1 + \frac{n_{t}}{n_{p}}} \right){p_{b}.}}}}$

Using (4), we can write (3) as

$\begin{matrix}{{\frac{n_{b}}{t} = {{\beta - {\alpha \; n_{b}} + {\gamma \; n_{b}^{2}}} = {{\gamma \left( {n_{b} - \lambda_{1}} \right)}\left( {n_{b} - \lambda_{2}} \right)}}},} & (5)\end{matrix}$

where λ₁ and λ₂ are introduced for convenience and denote the roots of

β−αn _(b) +γn _(b) ²=0.

Note that γ=β/(λ₁λ₂). The solution to (5) is found as

$\begin{matrix}{{n_{b}(t)} = {\lambda_{1} + {\frac{\lambda_{1}\left( {\lambda_{1} - \lambda_{2}} \right)}{{\lambda_{2}^{{\beta {({\frac{1}{\lambda_{1}} - \frac{1}{\lambda_{2}}})}}t}} - \lambda_{1}}.}}} & (6)\end{matrix}$

We should point out that (3) describes the change in the amount oftarget molecules, n_(b), captured by the probes in a single probe spotof the microarray. Similar equations, possibly with different values ofthe parameters n_(p), n_(t), p_(b), and p_(r), hold for other spots andother targets.

Estimating Parameters of the Model

The following is an outline of a procedure for estimation of theparameters. Ultimately, by observing the hybridization process, we wouldlike to obtain n_(t), n_(p), p_(b), and p_(r). However, we do not alwayshave direct access to n_(b)(t) in (6), but rather to y_(b)(t)=kn_(b)(t),where k denotes a transduction coefficient. In particular, we observe

$\begin{matrix}{{{y_{b}(t)} = {\lambda_{1}^{*} + \frac{\lambda_{1}^{*}\left( {\lambda_{1}^{*} - \lambda_{2}^{*}} \right)}{{\lambda_{2}^{*}^{\beta {({\frac{1}{\lambda_{1}^{*}} - \frac{1}{\lambda_{2}^{*}}})}}} - \lambda_{1}^{*}}}},} & (7)\end{matrix}$

where λ₁*=kλ₁, λ₂*=kλ₂, and β*=kβ.

For convenience, we also introduce

$\begin{matrix}{{\gamma^{*} = {\frac{\beta^{*}}{\lambda_{1}^{*}\lambda_{2}^{*}} = \frac{\gamma}{k}}},{\alpha^{*} = {{\gamma^{*}\left( {\lambda_{1}^{*} + \lambda_{2}^{*}} \right)} = {\alpha.}}}} & (8)\end{matrix}$

From (5), it follows that

$\begin{matrix}{\beta^{*} = {\frac{y_{b}}{t}_{t = 0}.}} & (9)\end{matrix}$

Assume, without a loss of generality, that λ₁* is the smaller and λ₂*the larger of the two, i.e., λ₁*=min(λ₁, λ₂), and λ₂*=max(λ₁,λ₂). From(7), we find the steady-state of y_(b)(t),

λ₁ *=limy _(b)(t), t→∞.  (10)

So, from (9) and (10) we can determine β* and λ₁*, two out of the threeparameters in (7). To find the remaining one, λ₂*, one needs to fit thecurve (7) to the experimental data.

Having determined β*, λ₁, and λ₂*, we use (8) to obtain α* and γ*. Then,we should use (4) to obtain p_(b), p_(r), n_(p), and n_(t) from α*, β*,and γ*. However, (4) gives us only 3 equations while there are 4unknowns that need to be determined. Therefore, we need at least 2different experiments to find all of the desired parameters. Assume thatthe arrays and the conditions in the two experiments are the same exceptfor the target amounts applied. Denote the target amounts by n_(t), andn_(t); on the other hand, p_(b) and p_(r) remain the same in the twoexperiments. Let the first experiment yield α₁*, β₁*, and γ₁*, and thesecond one yield α₂*, β₂*, and γ₂*(we note that γ₁*=γ₂*). Then it can beshown that

$\begin{matrix}{{p_{b} = \frac{{\beta_{1}^{*}\gamma_{1}^{*}} - {\beta_{2}^{*}\gamma_{2}^{*}}}{\alpha_{1}^{*} - \alpha_{2}^{*}}},{and}} & (11) \\{{p_{r} = {\alpha_{1}^{*} - p_{b} - {\frac{\beta_{1}^{*}\gamma_{1}^{*}}{p_{b}}.{Moreover}}}},} & (12) \\{{n_{p} = \frac{p_{b}}{k\; \gamma_{1}^{*}}},{and}} & (13) \\{{n_{t_{1}} = {\frac{\beta_{1}^{*}\gamma_{1}^{*}}{p_{b}^{2}}n_{p}}},{n_{t_{2}} = {\frac{\beta_{2}^{*}\gamma_{2}^{*}}{p_{b}^{2}}{n_{p}.}}}} & (14)\end{matrix}$

We note that quantities (13)-(14) are known within the transductioncoefficient k, where k=y_(b)(0)/n_(p). To find k and thus unambiguouslyquantify n_(p), nt_(t1), and n_(t2), we need to perform a calibrationexperiment (i.e., an experiment with a known amount of targets n_(t)).

Experiments were performed that were designed to test the validity ofthe proposed model and demonstrate the parameter estimation procedure.To this end, two DNA microarray experiments are performed. The custom8-by-9 arrays contain 25 mer probes printed in 3 different probedensities. The targets are Ambion mRNA Spikes, applied to the arrayswith different concentrations. The concentrations used in the twoexperiments are 80 ng/50 μl and 16 ng/50 μl. The signal measured in thefirst experiment, where 80 ng of the target is applied to the array, isshown in FIG. 22. The smooth line shown in the same figure representsthe fit obtained according to (7). In the second experiment, 16 ng ofthe target is applied to the array. The measured signal, and thecorresponding fit obtained according to (7), are both shown in FIG. 23.

Applying (11)-(14), we obtain

p _(b)=1.9×10⁻³ , p _(r)=2.99×10⁻⁵.

Furthermore, we find that

n _(t1) /n _(t2)=β₁*/β₂*=3.75.  (15)

Note that the above ratio is relatively close to its true value,80/16=5. Finally, assuming that one of the experiments is used forcalibration, we find that the value of the transduction coefficient isk=4.1×10⁻⁴, and that the number of probe molecules in the observed probespots is n_(p)=1.6×10⁻¹¹.

While the present invention has been particularly shown and describedwith reference to the preferred mode as illustrated in the drawing, itwill be understood by one skilled in the art that various changes indetail may be affected therein without departing from the spirit andscope of the invention as defined by the claims.

1-40. (canceled)
 41. A method comprising: (a) measuring the amount of 5or more amplicons corresponding to 5 or more different nucleotidesequences in a single fluid volume during or after multipleamplification cycles to determine amplicon amount-amplification cyclevalues, and (b) using the amplicon amount-amplification cycle values todetermine the presence or amount of the 5 or more nucleotide sequencesin a sample.
 42. The method of claim 41 wherein 20 or more ampliconscorresponding to 20 or more different nucleotide sequences are used todetermine the amount of 20 or more nucleotide sequences.
 43. The methodof claim 41 wherein 50 or more amplicons corresponding to 50 or moredifferent nucleotide sequences are used to determine the amount of 50 ormore nucleotide sequences.
 44. The method of claim 41 wherein themultiple amplification cycles comprise about 10-40 amplification cycles.45. The method of claim 41 wherein the amount of the 5 or more ampliconsis measured in real-time.
 46. The method of claim 41 wherein the amountof the 5 or more amplicons is measured by measuring the kinetics ofbinding of the amplicons to nucleic acid probes.
 47. The method of claim41 wherein the amount of the 5 or more amplicons is measured bymeasuring the quenching of fluorescence.
 48. A system comprising: (a) aPCR amplification reaction chamber capable of receiving: (i) a substratecomprising a surface with an array of nucleic acid probes atindependently addressable locations, and (ii) a fluid to be held contactwith the substrate, the fluid comprising a nucleic acid samplecomprising multiple nucleotide sequences, primers, and enzymes; (b) atemperature controller capable of carrying out multiple PCR temperaturephases and temperature cycles comprising: (i) a heating and coolingmodule for raising and lowering the temperature of the fluid and/or thesubstrate; and (ii) a temperature sensor; and (c) a detector capable ofdetecting light signals as a function of time from the independentlyaddressable locations on the substrate within the chamber at a specificphase or phases during or after a plurality of temperature cycles whilethe fluid is in contact with the substrate.
 49. The system of claim 48further comprising: (d) an analysis block comprising a computer andsoftware capable of determining the amounts of amplified productshybridized to the array of probes using the detected light as a functionof time, and of determining the amounts of multiple nucleotide sequencesin a sample using the amounts of amplified products determined during orafter a plurality of temperature cycles.
 50. The system of claim 48wherein the detector comprises a photodiode, a CCD array, or a CMOSarray.
 51. The system of claim 48 wherein the detector is in contactwith the substrate, and different areas of the detector correspond todifferent detectable locations.
 52. The system of claim 48 wherein thedetector is optically coupled to the substrate with lenses and/orwaveguides.
 53. The system of claim 48 wherein the heating and coolingmodule is capable of raising or lowering the temperature at a rate ofgreater than 5° C. per sec.
 54. The system of claim 48 wherein theheating and cooling module is capable of raising or lowering thetemperature at a rate of greater than 10° C. per sec.
 55. The system ofclaim 48 wherein the heating and cooling module is capable of raising orlowering the temperature at a rate of greater than 20° C. per sec. 56.The system of claim 48 wherein the temperature sensor is capable ofmeasuring temperature at an accuracy of 0.1° C.
 57. The system of claim48 wherein system comprises a microfluidic device having multiplearrays, each of the multiple arrays is in a chamber which can hold thefluid to be held in contact with the substrate.
 58. The system of claim57 wherein at least some of the multiple arrays are addressed withdifferent temperature profiles.
 59. The system of claim 57 wherein themicrofluidic device has about 4, 5, 6, 7, 8, 9, or 10 arrays.